Advancing puberty in female sheep: It’s all about fat and muscle · En esta vida no hay...

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Advancing puberty in female sheep: It’s all about fat and muscle by Cesar Augusto Rosales Nieto Bachelor of Science in Plant and Animal Science Master of Science in Animal Science This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia Faculty of Science School of Animal Biology 2013

Transcript of Advancing puberty in female sheep: It’s all about fat and muscle · En esta vida no hay...

Page 1: Advancing puberty in female sheep: It’s all about fat and muscle · En esta vida no hay accidentes, todo pasa por algo, cada quien es el arquitecto de su ... Esa es la historia

Advancing puberty in female sheep: It’s all about fat and muscle

by

Cesar Augusto Rosales Nieto

Bachelor of Science in Plant and Animal Science

Master of Science in Animal Science

This thesis is presented for the degree of Doctor of Philosophy of

The University of Western Australia

Faculty of Science

School of Animal Biology

2013

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Summary

The reproductive efficiency of the entire sheep flock could be improved if ewe lambs go

through puberty early and produce their first lamb at one year of age, rather than the

more ‘traditional’ two years. The onset of puberty is linked to the attainment of critical

body mass so the aim of the research described in this thesis was to test the general

hypothesis that, in Merino ewe lambs, accelerating the rate of growth and the

accumulation of muscle and fat will advance puberty and improve reproductive

success. A secondary hypothesis tested was that the effects of accelerated growth on

reproduction are mediated by changes in the concentrations of follistatin and leptin.

We studied the statistical relationships between reproductive performance and a

variety of measures of growth and body composition: phenotypic values for depth of

eye muscle (EMD) and depth of fat (FAT) and genotypic values (Australian Sheep

Breeding Values) for similar measures made post-weaning, live weight (PWT) and

depths of eye muscle (PEMD) and depth of fat (PFAT). First oestrus was detected with

vasectomized rams and then entire rams as the ewes progressed from 6 to 10 months

of age.

The general hypothesis was supported by observations in several experiments

involving over 840 Merino ewe lambs over two seasons. Age at first oestrus decreased

with increases in values for PWT. The proportion of ewe lambs that achieved puberty

was positively related to increases in the values for EMD, FAT or PEMD, PFAT or

PWT. Ewe lambs that were heavier at the start of mating were more fertile and had a

higher reproductive rate. Fertility and reproductive rate were positively correlated with

values for EMD, FAT, PWT, PEMD, PFAT.

However, many ewe lambs attained puberty (first oestrus) but then failed to

conceive, so fertility and reproductive rate varied significantly in ewe lambs of similar

live weight at the start of mating, suggesting that the outcome is determined by other

factors. We tested the importance of liveweight change during mating. Females that

weighed 40 kg at the start of the mating period were assigned to dietary treatments that

targeted low (40 kg) or high (45 kg) live weights during mating. Females that were

assigned to target 45 kg were more fertile and had a higher reproductive rate than

females that were targeting 40 kg.

We assessed the roles of some hypothetical endocrine mediators between body

tissues and the reproductive system. The link from adipose tissue is obviously leptin

but muscle hormones associated with reproduction have not been clearly identified.

One possibility is follistatin. We therefore measured blood concentrations of leptin and

follistatin so we could test whether they were related to production traits and

reproductive performance. Leptin concentration was positively correlated with values

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for EMD, PEMD, FAT, PFAT and PWT, whereas, in general, follistatin concentration

was negatively correlated to the same traits. Leptin concentration was also positively

related to age and live weight at first oestrus, the proportion of females that attained

puberty, and fertility and reproductive rate. There was also evidence that leptin

produced by intramuscular fat plays a role in the onset of puberty. By contrast,

follistatin concentration was negatively related with live weight at first oestrus, fertility

and reproductive rate.

We can conclude that, in ewe lambs mated at about 8 months of age, increasing

live weight during mating will increase fertility and reproductive rate, and this can be

achieved by the use of higher breeding values for accumulation of muscle and fat, and

therefore growth. These relationships, certainly for adipose tissue and perhaps also for

muscle, appear to involve a stimulatory role played by leptin on the reproductive control

centres. The regulatory role of muscle might also be mediated by follistatin, but this

hormone appears to be inhibitory and thus has to be withdrawn to allow for puberty to

proceed and fertility to be expressed.

For the sheepmeat industry, it is clear that strategies for genetic improvement of

body composition, particularly with a view to more rapid muscle (carcase)

accumulation, will very likely also advance puberty and improve the overall

reproduction rate of the flock. The goal of strong reproductive performance in the first

year of life is within reach, as are a major step-up in the lifetime reproductive

performance of each ewe.

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Table of contents Summary i

Table of contents iii

Dedicatoria iv

Acknowledgements v

Statement of contribution viii

Publications ix

General introduction 1

Literature review 5

Chapter 1 Selection for superior growth advances the onset of

puberty and increases reproductive performance in ewe

lambs (animal 7, 990 - 997.)

19

Chapter 2 Ewe lambs with higher breeding values for growth

achieve higher reproductive performance when mated

at age 8 months (Theriogenology 80, 427-435)

38

Chapter 3 Prepubertal growth and muscle and fat accumulation in

male and female sheep – relationships with metabolic

hormone concentrations, timing of puberty, and

reproductive performance (Domestic Animal

Endocrinology, in preparation)

58

Chapter 4 Roles of liveweight change during mating and muscle

accumulation on puberty and fertility in ewe lambs

(Animal Production Science, in preparation)

76

Chapter 5 Relationships among body composition, circulating

concentrations of leptin and follistatin, and the onset of

puberty and fertility in female sheep (Journal of Animal

Science, in preparation)

92

General discussion 111

References (general introduction, literature review and general discussion) 116

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Dedicatoria

“Being deeply loved by someone gives you strength, while loving someone deeply

gives you courage” (Lao Tzu)

En esta vida no hay accidentes, todo pasa por algo, cada quien es el arquitecto de su

propio destino! Esa es la historia de mi vida! Esa es la historia de nuestras vidas!

Dependerá de nosotros si somos capaces de descifrar ese acertijo para descubrir

nuestro potencial. Las cosas negativas que en su momento vivimos, nos sirvieron para

salir adelante, fortalecernos y darnos cuenta de que el mundo no termina ahí; que la

vida sigue y que dependerá de nosotros hasta donde queremos llegar. Cada fracaso y

cada logro que hemos vivido han fortalecido la relación, ha fortalecido nuestro

carácter. Gracias al ranger por no conocer al comandante Peña. Gracias a eso, hoy

estamos aquí, hoy culminamos una etapa más en nuestra vida. Una etapa que no

cambiaría por nada en el mundo!

Por eso, tenemos que hacer que cada día cuente como si fuera el último. No

sabemos que nos depara el destino. No sabemos cuánto tiempo más vamos a estar

en este mundo. Hay que vivir y gozar la vida! El ayer ya es historia; el futuro es muy

incierto, pero el día de hoy es un regalo; por eso se llama presente y hay que

disfrutarlo como si fuera el último día (Modificado de Master Oogway).

Mónica, Natalia y Romina, esta va dedicada a ustedes; esto es la culminación

de cuatro años de estudio, de preparación y sacrificios. Ustedes son la razón de mí

existir y por lo cual yo me esfuerzo cada día más. Yo estuve tan inmerso en mi trabajo

que me ausente literalmente 3 años de la casa; sin embargo te tuve a ti Moniquiu y tus

hijas te tuvieron a ti; tuviste la casta y el aplomo de sacar esto adelante; casa, trabajo y

lo más importante a las hijas! Los éxitos y reconocimientos que tuve como estudiante y

los que tendré como profesional, fueron y serán logros para todos nosotros; ya que

somos una familia! Todo esto no hubiera sido posible sin su apoyo! Muchas gracias

por aguantar ese ogro que atacaba de repente, pero más importante, por ese apoyo

incondicional, por sus palabras de aliento que nunca me faltaron.

También va dedicada a los que no estuvieron con nosotros este tiempo, mi

mama, hermanas y sobrinos, sin embargo siempre estuvieron al pendiente de nosotros

y que desde lejos nos daban su apoyo incondicional.

Este tiempo que estuvimos fuera de México y que Australia nos cobijó como

nuestro nuevo hogar siempre tuvimos un ángel que nos cuidó. Tu y yo sabemos bien

quién es; gracias por estar siempre cuidándonos!

Mor’du es por ustedes y para ustedes!!

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Acknowledgements

“Choose a job you love, and you will never have to work a day in your life” (Confucius)

“Research is what I'm doing when I don't know what I'm doing” (Wernher von Braun)

“Learn from yesterday, live for today, hope for tomorrow. The important thing is not to

stop questioning” (Albert Einstein)

“No amount of experimentation can ever prove me right; a single experiment can prove

me wrong” (Albert Einstein) To my supervisors “The way a team plays as a whole determines its success. You may have the greatest

bunch of individual stars in the world, but if they don't play together, the club won't be

worth a dime” (Babe Ruth)

“I hear and I forget. I see and I remember. I do and I understand” (Confucius)

“If I am walking with two other men, each of them will serve as my teacher. I will pick

out the good points of the one and imitate them and the bad points of the other and

correct them in myself” (Confucius)

Thank you for believing in me, thank you for giving me this opportunity and thank you

for accepted to be my supervisors. It was such a great pleasure to work with you. This

was an enjoyable learning experience for me and your guidance taught me many

valuable lessons through this PhD process that will help me in my career. Thanks for

being so detailed in so many aspects, particularly in our papers. I learned to work

professionally and to become a better listener and a fast learner. It was very

challenging to combine different points of view; three great minds that think so

differently. You were very patient with me and you took your time to explain my tasks

and help me to comprehend the work that I needed to accomplish. Thank you for your

support and those words of encouragement that you had for me every time. All my

achievements as a person/student reflect the dedication and contribution that you had

to me. I was very fortunate to have you all.

Here, I would like to extend my thanks and gratitude to Dominique Blache.

Despite the fact that he was not my supervisor, he acted like one. His door was always

open for me and in countless occasions he responded my questions and solved my

doubts.

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To my team

“Unity is strength... when there is teamwork and collaboration, wonderful things can be

achieved” (Mattie Stepanek)

“When a team outgrows individual performance and learns team confidence,

excellence becomes a reality” (Joe Paterno)

“Success depends upon previous preparation, and without such preparation there is

sure to be failure” (Confucius)

Jan and Claire: OMG, seriously this would not have been possible without your help.

You are an important part of my achievements as a student. How many sheep we bled;

how many sheep we weighed; how many trips we had to Medina, to Ridgefield, to

Moojepin. In this group, we always had long sessions and interminable work but we

always finished the job! Thank you for your assistance, guidance, patience and

friendship! Thank you for taking care of my experiment while I had a broken leg.

Margaret Blackberry, thank you for your help during my time in the lab – without your

guidance my samples would have been a disaster. As well, I want to extend my

gratitude to Duncan, Sarah, Beth and Ox, who were always keen to help!

Thank you Hamish and Dave Thompson for your hospitality and help during my

experiment in Moojepin.

To my sponsors “Education is what remains after one has forgotten what one has learned in

school” (Albert Einstein)

Thank you for believing in me. I am fortunate to have had the financial support provided

by CONACyT (the Mexican National Council for Science and Technology), the

Department of Agriculture and Food of Western Australia, the Cooperative Research

Centre for Sheep Industry Innovation and the UWA School of Animal Biology.

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To Wendy, Alan, Rose, Graeme, Jackie, Nisha, Mark and Thommo

“Some people come into our lives and quickly go. Some people move our souls to

dance. They awaken us to a new understanding with the passing whisper of their

wisdom. Some people make the sky more beautiful to gaze upon. They stay in our lives

for awhile, leave footprints on our hearts, and we are never, ever the same” (Flavia Weedn)

Thank you guys for your support during all this time. This would have been a very

different history.

To our Mexican family in Perth

Jior, Aletzin, Mariana, Lalo, los Cucos, Susana, Jorge, Lasha and Luis. As Mexicans,

we always look for coexistance and we were very lucky because we found a family in

you!! For the parties, Christmas, New Year’s we celebrated together! We always had a

dish, a beer and history to share! Thanks to your friendship we were not homesick; our

Mexico was here with you!

To my post grad fellows Dr Machado, Mikaela, Xixi, Yongjuan, Kels, Stacey, Trina, Sam, Steph, Jo, Kirrin, Joe,

Umar and my CRC mates. Thank you for sharing a cup of tea and enlightening the

room with jokes and comments. Thank you for your friendship! We are the network of

the future; let’s keep in touch!! Thank you Mikaela for being part of the noisy room!!

To myself “The great man is he who does not lose his child's-heart” (Mencius) “You have to believe in yourself” (Sun Tzu) “When you are content to be simply yourself and don't compare or compete, everybody

will respect you” (Lao Tzu) “Try not to become a man of success, but rather try to become a man of value” (Albert Einstein) “Knowing others is wisdom, knowing yourself is Enlightenment” (Lao Tzu) “If you can't explain it simply, you don't understand it well enough” (Albert Einstein) “The only source of knowledge is experience. You have to learn the rules of the game.

And then you have to play better than anyone else” (Albert Einstein)

Always be yourself, help others, always remember who you are and from where you are coming from. Be a good husband and excellent dad for Natalia and Romina.

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Statement of contribution

The work presented in this thesis is the original work of the author. This thesis contains

published work and/or work submitted for publication, some of which has been co-

authored. The experimental work, statistical analyses and manuscript preparation was

carried out by the author of this thesis and discussed in extensive detail with the

supervisors Mr Mark Ferguson, Dr Andrew Thompson and Prof Graeme Martin before

submission to the journal. All experimental work required the help of volunteers and Mr

Mark Ferguson, Dr Andrew Thompson, Prof Graeme Martin, Dr Mark Hedger, Ms Jan

Briegel, Ms Claire Macleay, Mr Duncan Wood and Mr Hamish Thompson contributed

significantly to the collection of data, or analyses of data, and are co-authors for some

manuscripts in recognition of their contribution.

Cesar Augusto Rosales Nieto August 2013

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Publications arising from work in this thesis

Journal articles (peer reviewed)

Rosales Nieto C.A., Ferguson M.B., Macleay C.A., Briegel J.R., Martin G.B., Thompson A.N. Selection for superior growth advances the onset of puberty and

increases reproductive performance in ewe lambs. animal 7, 990-997.

Chapter 1

C.A. Rosales Nieto, M.B. Ferguson, C.A. Macleay, J.R. Briegel, D.A. Wood, G.B. Martin, A.N. Thompson. Increasing genetic potential for growth improves the

reproductive performance of ewe lambs. Theriogenology 80, 427-435.

Chapter 2

Conference proceedings

C.A. Rosales Nieto, M.B. Ferguson, C. Macleay, J. Briegel, A. Thompson, G.B. Martin. 2010. Growth rate, muscling and reproduction in Merino ewe lambs. Animal

Production Science (50) vii.

Chapter 1

C.A. Rosales Nieto, M.B. Ferguson, C. Macleay, J. Briegel, G.B. Martin, A. N. Thompson. 2011. Selection for superior growth advances the onset of puberty in

Merino ewes. Proceedings of the Association for the Advancement of Animal Breeding

and Genetics, 19, 303-306.

Chapter 1

C.A. Rosales Nieto, M.B. Ferguson, C.A. Macleay, J.R. Briegel, M.P. Hedger, G.B. Martin, A.N. Thompson. 2012. Follistatin, muscle development, puberty and fertility in

ewe lambs. Proceedings of the Annual Meeting of the European Federation of Animal

Science, 18, 284.

Chapter 5

Media coverage

Early breeding to boost flock sizes. Ag in Focus, Department of Agriculture and Food of

Western

Australia. http://agric.wa.gov.au/objtwr/imported_assets/aboutus/pubns/agif_wa_summ

er_2011-12.pdf

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Can joining Merino ewe earlier accelerate the flock rebuild? CRC for Sheep Industry

Innovation. http://www.sheepcrc.org.au/information/news.php

Can joining Merino ewe earlier accelerate the flock

rebuild? http://sj.farmonline.com.au/news/state/livestock/sheep/can-earlier-joining-

accelerate-flock-rebuilding/2539077.aspx

Will early joining help increase flock

number? http://www.therural.com.au/news/local/news/general/will-early-joining-help-

increase-flock-numbers/2538474.aspx

Success with joining lambs earlier. Farming

Ahead. http://www.kondiningroup.com.au/StoryView.asp?StoryID=8682432

Promise in early

joining http://tascountry.realviewtechnologies.com/?iid=62566&startpage=page000002

2

Merino ewes accelerate flock rebuild? http://www.silobreaker.com/merino-ewes-

accelerate-flock-rebuild-5_2265658601555099736

Finding the best way to build flock numbers. Esperance Express, general news page

11. Circulation 3,500. Date 02/05/2012.

Research focuses on joining Merino ewes at earlier age. Narromine

News. http://www.narrominenewsonline.com.au/news/local/news/rural/research-

focuses-on-joining-merino-ewes-at-earlier-age/2542161.aspx

Can joining Merino ewe earlier accelerate the flock rebuild? Southern

Weekly http://www.southernweekly.com.au/news/local/news/general/can-joining-

merino-ewes-earlier-accelerate-the-flock-rebuild/2544534.aspx

Joining ewes earlier a possibility. Bombala Times, general news page 12. Date

16/05/2012.

Can joining Merino ewes earlier accelerate the flock rebuild? Border News, general

news page 5. Date 7/05/2012.

Seeking ways to grow flocks. Country News inserts, Shepparton Vic. General news

page 49. Regional circulation 49,616. Date 30/04/2012.

Feeding for better fertility. MLA. Meat and Livestock

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Australia. http://www.mla.com.au/Publications-tools-and-events/Industry-

news/Feeding-for-better-

fertility#hp=highlight2&article=Feeding%20for%20better%20fertility

Vale la pena usar el servicio temprano a

corderas? http://www.woolpatagonia.com.ar/noticias/internacionales/notas/2012/mayo/

22/corderas.htm

PhD project sets new direction for sheep breeding research. The UWA Institute of

Agriculture. IOA April 2013.

Early mating defies tradition. The Countryman. June 2013.

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General introduction

Australia is the principal producer of export lamb and mutton for the world (Meat

and Livestock Australia, 2012), but the Australian sheep industry cannot meet current

demands for export lamb. Therefore, it is necessary to improve the reproductive

efficiency of the sheep flocks and this has renewed attention on the breeding of young

ewes in their first year of life (Martin, et al. 2009; Ferguson, et al. 2011). Mating ewes

as lambs offers the opportunity to increase the number of lambs on the ground and

therefore exploit potential advantages in sheep breeding and selection programs

through the reduction of generation interval. In addition, it has been demonstrated that

if a ewe lamb can rear lamb(s) successfully to weaning there is the potential to

increase profitability (Young, et al. 2013) and lifetime reproductive performance

(Kenyon, et al. 2011). However, this concept is not well accepted by producers due to a

variety of perceptions: increase on-farm costs, more labour required, poor and variable

reproductive performance, an increase in feed demand, greater live weight and body

condition score targets at a young age, low survival of new-born progeny, tendency for

progeny to be lighter at birth and weaning, and greater death rates of ewe lambs

(reviewed by Kenyon, et al. 2013).

Given these potential constraints, we need specific guidelines for the

reproductive management of young animals so producers can maximize the chances

of success. For example, for young ewes raised under Australian conditions, producers

need to know the live weight, liveweight change, body condition and age at which

puberty is reached.

The timing of puberty is the outcome of dynamic interactions among several

genetic and environmental factors (reviewed by Dýrmundsson 1981) and generally

occurs in ewe lambs when they attain 50 to 70% of their expected mature body mass

(Hafez, 1952; Dýrmundsson 1973). If growth is restricted during early life, young ewes

will remain pre-pubertal until the required proportion of mature body mass is reached

(reviewed by Foster, et al. 1985) so slowly-growing lambs achieve puberty later than

rapidly-growing lambs (Boulanouar, et al. 1995). This relationship between growth rate

and puberty explains the earlier puberty in ewes raised as singles compared to those

raised as twins across a range of breeds (Southam, et al. 1971) – single-reared lambs

grow faster to weaning and can remain heavier until 12 months of age despite post-

weaning compensatory growth in twin-reared progeny (Thompson, et al. 2011). On the

same basis, we would expect puberty to occur earlier in ewe lambs with higher

breeding values for rapid growth.

After puberty, there are positive correlations between weaning weight, growth

and reproductive performance in young female sheep (Barlow and Hodges, 1976;

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Gaskins et al., 2005). In addition, fertility and reproductive rate in young ewes are

related to live weight at mating (McGuirk, et al. 1968). Therefore, we would also expect

that ewe lambs with higher growth rates due to better environmental conditions or

higher breeding values for growth would be more fertile and have higher reproductive

rates than ewes with lower growth rates.

However, fertility and reproductive rate vary significantly in ewe lambs of similar

live weight at the start of mating, suggesting that the outcome is determined by other

factors such as liveweight change (LWC). An effect of LWC during mating on

reproductive success could be explained by the ‘acute’ metabolic effect in which short-

term changes in nutrition affect ovarian function, independently of changes in live

weight, or the ‘dynamic’ effect associated with changes in live weight before conception

(reviewed by Scaramuzzi et al. 2006). However, it is not known if the ‘dynamic’ effect

simply reflects weight gains during mating that lead to the ewes being heavier when

mated, or if liveweight change itself has some effect on fertility and reproductive rate in

addition to those associated with correlated changes in absolute live weight.

Regardless of the mechanism, it is expected that improving the nutrition of ewe lambs

so they gain weight during mating will increase their fertility and reproductive rates.

In addition to the effects of growth rate and live weight at mating, body

composition might also be related to the timing of first oestrus, fertility, and reproductive

rate. In mature Merino ewes, fertility and reproductive rate are known to be better for

genotypes with higher breeding values for percentages of muscle and fat that they had

when they were yearlings (Ferguson, et al. 2007; 2010). Furthermore, phenotypic

enhancement of muscle mass and fatness, as assessed through condition score, are

known to increase fertility and reproductive rate in ewes mated to lamb at one or two

years of age (Malau-Aduli, et al. 2007). It is not clear whether this also applies to ewe

lambs and their development of reproductive capability but, overall, it appears that ewe

lambs that accumulate fat and muscle more rapidly will achieve puberty earlier, be

more fertile and have a higher reproductive rate when mated at 8 or 9 months than

their counterparts.

All of the observations cited above show that the metabolic status of the animal

is involved in the regulation of sexual maturation and reproductive performance,

presumably due to the effects of physiological signals from metabolic tissues on the

reproductive axis (review: Martin, et al. 2008). To understand the proposed links

between the rates of muscle and fat accumulation and reproduction in young sheep,

we need to search for physiological links between reproductive control systems and the

muscle and adipose tissues. The metabolites and hormones involved in these

processes include growth hormone (GH), insulin-like growth factor (IGF-I), insulin,

ghrelin, leptin, follistatin, glucose and non-esterified fatty acids (NEFA). The primary

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candidate from adipose tissue is leptin, a potent regulator of nutrition, metabolism and

reproductive endocrinology (Blache, et al. 2000). The role of leptin in puberty has been

extensively examined in a variety of species, including monogastrics (Ahima, et al.

1997; Cheung, et al. 1997; Kiess, et al. 1998; Barb, et al. 2004; Maciel, et al. 2004) and

ruminants (Foster and Nagatani, 1999; Chilliard, et al. 2005).

With respect to muscle, endocrine factors associated with reproductive

performance have not been clearly identified, but IGF-I and follistatin are possibilities.

IGF-I controls muscle growth (reviewed by Oksbjerg, et al. 2004) and circulating IGF-I

concentration has been associated with the onset of puberty in ewe lambs (Roberts, et

al. 1990). Follistatin plays an important role in muscle growth and development during

fetal development and early neonatal life (McFarlane, et al. 2002). In mice, follistatin

overexpression increases muscle weight and mass (Lee and McPherron 2001; Lee

2007; Gilson, et al. 2009), corroborating the earlier work of Matzuk, et al. (1995) who

had reported retarded growth, reduced muscle mass, bone deficiencies, and early

postnatal death in follistatin-deficient mice. Therefore we would expect follistain

concentrations to be high during muscle growth and development and higher in ewe

lambs selected for rapid muscle accumulation. The role of follistatin in reproduction is

less clear. In the hypothalamic-pituitary axis, it has no effect on GnRH secretory

patterns in sheep (Padmanabhan, et al. 2002) but it inhibits FSH secretion in rodents

(Ueno, et al. 1987). In the ovary, it is synthesized by granulosa cells and helps in the

mechanism of follicular selection during the period leading to ovulation (Michel, et al.

1990; Nakatani, et al. 1991; Xiao, et al. 1992). During ovulation, follistatin seems to

bind to activin and control its actions, an important part of the ovulatory process

(Nakamura, et al. 1990; Newton, et al. 2002). Overall, the importance of follistatin in

reproduction was demonstrated in mice: first, deletion of follistatin in adult granulosa

cells alters follicle and oocyte maturation, reduces fertility and terminates ovarian

activity (Jorgez, et al. 2004); second, deletion of one isoform of follistatin reduces litter

size and leads to early cessation of reproduction (Kimura, et al. 2010). Overall, the

situation is not at all clear, but it seems likely that changes in the circulating

concentration of follistatin will influence reproductive function (review: Knight, 1996).

Therefore, the aim of the research described in this thesis was to test the

general hypothesis that, in Merino ewe lambs, the rate of growth and accumulation of

muscle and fat will positively influence the concentration of follistatin and leptin, thus,

the age and live weight at puberty and the reproductive success. The aim was study in

detail Merino ewes with variation in growth and accretion of muscle and fat and

establishes whether these factors were related to reproductive success at young ages.

The following specific hypotheses were tested:

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1. Merino ewe lambs with higher values for the rate of growth, and

accumulation of muscle and fat will be younger at first oestrus and a greater

proportion of them will attain puberty than their counterparts.

2. Merino ewe lambs with higher values for the rate of growth, and

accumulation of muscle and fat will be heavier at first oestrus and a greater

proportion of them will attain puberty than their counterparts.

3. Higher value for the rate of growth and accumulation of muscle and fat will

allow a greater proportion of Merino ewe lambs to conceive with a greater

reproductive rate, compared to ewe lambs with lower values for these traits.

4. The effect of the higher values for the rate of growth and accumulation of

muscle and fat on reproductive performance will be greater in ewe lambs that

are lighter at start of mating, or growing less during mating, compared to ewe

lambs with lower values for the rate of accumulation of muscle and fat.

5. Differences in the rate of growth and accumulation of muscle and fat in ewe

and ram lambs will lead to differences in the circulating concentrations of

metabolites and metabolic hormones that are linked to the control of the

reproductive system, and will therefore be related to the onset of puberty and

the success of reproduction.

6. Higher values for the rate of growth and accumulation of muscle and fat will

influence age and live weight at first oestrus, and fertility and reproductive

rate, via effects on the concentrations of leptin and follistatin.

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Literature Review

The Australian sheep industry is showing an interest in improving carcass and

reproductive traits due to the relative increases in prices for meat and the sustained low

prices for wool. In particular, the ability of the lamb industry to meet projected demand

requires drastic improvements in weaning rates from Merino ewes. Mating replacement

ewes to lamb at age 12-14 months rather than 2 years would significantly increase the

profitability of lamb production systems based on crossbred ewes, but the benefit

gained by early mating depends on the reproduction rate achieved (Young, et al.

2010). This review will describe the Australian sheep industry and its needs, and

outline the external and internal factors that affect the onset of puberty and the

reproductive performance in young females. This topic encompasses the physiology of

adipose and muscle tissue and possible links with the reproductive axis. Finally, there

will be an assessment of the impact of genetic selection using Australian Sheep

Breeding Values (ASBVs) on important economic traits in sheep that are link meat

production to reproduction.

The Australian sheep industry

In 1970, the sheep population in Australia was just under 180 million but, by 2010, it

had decreased to 68 million; recently, it has increased slightly to 73 million (Figure 1).

Most sheep are located in New South Wales (26.8 million), followed by Victoria (15.2

million), Western Australia (14 million), South Australia (11 million), Queensland (3.6

million) and Tasmania (2.3 million). Around 75% of sheep in Australia are Merinos

(90% in WA).

Figure 1. Changes in the Australian sheep population. Australian Bureau of Statistics (2013b).

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The sheep industry used to be focussed on the production of one commodity,

wool, so, until the last decade, most producers retained castrated males (wethers) as a

significant portion of their flock. With the decline in wool profits, wethers lost value so

producers now keep only female progeny and the males are sold as lamb or exported

(when they are 7-24 months old) to Asia, Africa and North America (Meat and

Livestock Australia, 2012). Producers have also responded to the changing markets by

increasing the production of lambs specifically bred for slaughter, rather than just for

wool production. This has led to a change in the composition of the national flock as

producers crossed Merino ewes with other breeds to produce prime lamb.

Another factor troubling the sheep industry is an increasing concern about

methane emissions (Hegarty 2008). Importantly, with respect to the theme of this

thesis, sheep that are not reproducing are only producing methane, so there is

pressure to reduce the length of infertile periods, thus decreasing ‘emissions intensity’

(units of methane per unit of product).

These needs, to produce more lambs, to produce them faster, and to reduce

emissions intensity, are driving a renewed interest in improving the reproductive

efficiency of the sheep flock. The focal points are ovulation rate (number of eggs

released by an animal that ovulates), fertility at mating, embryo mortality, postnatal

mortality, and delayed puberty.

Reproduction in female sheep General concepts

The reproductive system is primarily controlled by the activity of gonadotrophin-

releasing hormone (GnRH) neurons in the brain. GnRH is synthesized by these

neurons and transported along their axons to the median eminence where it is released

into the hypophyseal portal system. The activity of the GnRH neurons is synchronised

by an unknown mechanism, so the GnRH is release in pulses. The frequency of the

GnRH pulses is the major determinant of reproduction: by increasing pulse frequency,

the brain activates the ovary, and by decreasing pulse frequency it allows ovarian

activity to subside.

The hypophyseal portal system transports the GnRH pulses to the anterior

pituitary gland, where they regulate the synthesis and release of gonadotrophins,

luteinizing hormone (LH) and follicle stimulating hormone (FSH), the two major

hormones that control gonadal activity (Figure 2). The pattern of secretion of LH is also

pulsatile, mimicking the GnRH pulses, but FSH secretion is continuous. The

gonadotrophins are transported to the ovaries where they promote the synthesis and

secretion of inhibin and the sex steroids, primarily oestradiol and progesterone. There

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are two feedback loops, the first involving the follicle(s) and the pituitary gland, and the

second involving the follicles, the CL, and the brain. Both loops involve synergistic

interactions. High progesterone concentrations produced by the corpus luteum reduce

GnRH pulse frequency, leading to reduced oestradiol production by the follicles. Low

progesterone concentrations during the follicular phase allow an increase in GnRH

pulse frequency, so oestradiol production by the dominant follicle will increase and elicit

oestrus, the LH surge (positive feedback), and ovulation. Oestradiol and inhibin, both of

which are follicular products, will reduce FSH concentrations, thus reducing follicular

growth.

Figure 2. The major endocrine pathways that control ovarian function in the female, illustrating

the pituitary control of ovarian production of inhibin and sex steroids, and the roles of these

hormones in negative feedback.

The balance of the hypothalamic-pituitary-ovarian axis is also influenced by a range of

external factors, most acting at brain level (photoperiod, socio-sexual signals, nutrition),

although metabolic and nutritional factors also act directly on the ovary (Fig 3).

Puberty

During the pre-pubertal phase, when the ewe lamb does not show ovulatory activity,

GnRH/LH pulse frequency is low (Bindon and Turner 1974; Foster, et al. 1975a; 1975b;

Huffman, et al. 1987). The low pulse frequency does not provide the sufficient stimulus

for follicular development, so the young ewe remains in anovulatory state (Foster et al.,

1986). The brain of the lamb is capable of generating the high frequency of GnRH/LH

pulses needed for inducing ovulation, and can do so when the ovaries are removed,

but the hypothalamus is very responsive to negative feedback by oestradiol, even in

the absence of progesterone (Fig. 2), so GnRH secretion is inhibited (Foster, et al. 7

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1986). Circulating concentrations of FSH reach the adult range by 10-12 weeks of age

(Fitzgerald and Butler 1982; Foster, et al. 1975a), although a rise in FSH concentration

during the peri-pubertal period has been reported (Ryan, et al. 1991).

Figure 3. Schematic summary of the potential relationships among the endocrine and neural

inputs into the systems that control the reproductive system and mediate the responses to

change in metabolic status. Stimulation of the secretion of IGF-I by growth hormone (GH) acting

on the liver is omitted for clarity. Broken lines indicate hypothetical but unproven pathways.

Modified after Martin, et al. (2010).

There is a gradual 3-4-fold increase in GnRH/LH pulse frequency over the week

before first ovulation (Huffman, et al. 1987), because there is a decrease in the ability

of oestradiol to inhibit GnRH secretion (reviewed by Karsch 1987). The high GnRH

pulse frequency initiates a follicular phase that culminates in ovulation (Foster and

Karsch 1975). The first ovulation is often not accompanied by behavioural oestrus

(known a ‘silent’ ovulation), but the second ovulation usually is (Foote, et al. 1970;

Ryan, et al. 1991). First ovulation and first oestrus thus typically occur within 2 to 3

weeks of each other and several patterns have been reported with respect to the

temporal relationship between them (Ryan, et al. 1991).

The corpus luteum produced by the first ovulation can also be short-lived,

leading to a short luteal phase and a second ovulation 2-8 days after the first (Ryan, et

al. 1991; Baird, 1992). Both the failure of oestrus and the short cycle are caused by the

lack of circulating progesterone in the period leading up to the first ovulation, so the

presence of progesterone during the first normal luteal phase generally guarantees

oestrus (Ryan, et al. 1991; Baird, 1992; Keisler, et al. 1985). Thereafter, progesterone 8

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assumes the role of ‘cycle controller’ and determines the interval between ovulations

(Thimonier 1979).

First oestrus is an indication of attained of sexual maturation and occurs in ewe

lambs when they have attained 50-70% of mature body weight (Hafez, 1952;

Dýrmundsson 1973). However, the onset of puberty varies from individual to individual,

from flock to flock within breed, and from location to location, and the timing is affected

by a variety of internal and external factors:

a) Season of birth can affect age and body weight at puberty (Watson and Gamble

1961; Wiggins, et al. 1970; Hawker and Kennedy 1978);

b) Local environmental factors, such as environmental temperature, which is closely

associated with light changes in natural conditions, may contribute to the variation

of the onset of puberty. Ewe lambs attain puberty more slowly in tropical regions

than in temperate regions (Moule 1950). Interestingly, the delay of puberty under

extreme temperatures is attributed to a slower growth rate (Hafez, 1964).

c) Social cues, such as the sudden introduction of mature rams will advance puberty

(Oldham and Grey 1984).

d) Genetic factors – there are breed differences and variation among individuals

within the same breed in the age and body weight at first oestrus (Hafez, 1952;

Dickerson and Laster 1975; Quirke, et al. 1985; Fogarty, et al. 2007).

The importance of body weight shows the relevance of growth rates. Failure to

grow to the appropriate body size will leads to maintenance of the anovulatory

condition (Hafez, 1952). Puberty is earlier in ewes raised as a single than in those

raised as a twin, across a range of breeds (Southam, et al. 1971). Modification of

growth by manipulation of nutrition will influence age and body weight at puberty. Thus,

severe under-nutrition, or restriction of the intake of protein or energy, delays the onset

of puberty in the ewe lamb (Foster and Olster, 1985; Boulanouar, et al. 1995). It is

arguable that such effects are due to the plane of nutrition per se or due to the growth

rate of the animal as result of the plane of nutrition.

Seasonality

Reproductive seasonality is common to many species that inhabit the non-equatorial

regions of the world. It is an adaptation to the annual cycles in temperature and food

availability and it evolved to ensure birth at the optimal time of the year for lactation and

for the growth of the new-born. Most genotypes of sheep exhibit ovarian activity

primarily between late summer and early winter. During the remainder of the year, they

are anoestrous, as indicated by the almost total absence of sexual behavior. These

changes in the annual cycles can profoundly affect the age at puberty in ewe lambs

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(Foster, 1981; 1983; Ebling and Foster, 1988). In general, the sexual season will be

longest in sheep from topical regions and shortest in sheep from sub-polar regions

(Hulet, et al. 1974a; 1974b; Chemineau, et al. 1992). Some flocks located in tropical

regions have been reported to show little seasonality (Jackson, et al. 1990;

Chemineau, et al. 2004). Seasonal breeding has a long evolutionary history but is a

major limitation to reproductive efficiency in industrial systems.

Nutrition-reproduction interactions

There is a strong association between nutrition and reproduction, ranging from minor

changes in ovulation rate or frequency to cessation of breeding or extension of the

anoestrus season if nutrition is very restricted. Undernutrition during early stages of

development, either before or after birth, can induce a reduction in the lifetime

reproductive capacity of the female offspring (Gunn, et al. 1995; Rhind, et al. 1998).

Undernutrition disrupts the hypothalamic GnRH pulse generator and thus the secretion

of LH and FSH (Dunn and Moss 1992; Schillo 1992; Mani, et al. 1996) and decreases

the release of LH during the luteal phase of the oestrous cycle in sheep and heifers

(Mackey, et al. 1999; Kiyma, et al. 2004). At the level of the ovary, the outcome is

reduced follicular growth (Bergfeld, et al. 1994), causing atresia in those follicles that

begin development during nutrient restriction (Diskin, et al. 2003), leading to a

reduction in the percentage of females that ovulate or ovulation rate (Killeen 1982;

Abecia, et al. 1997; Mackey, et al. 1999). On the other hand, nutritional

supplementation increases the concentration of progesterone and the amplitude and

frequency of the LH pulses (Imakawa, et al. 1983 and 1986; Rhind, et al. 1985), and

improves ovulation rate (Viñoles, et al. 2005 and 2009).

In addition, dietary restriction will reduce conception rate (reviewed by Abecia,

et al. 2006) and, during pregnancy, compromise the development of the embryo

(Lozano, et al. 2003; Luther, et al. 2007) and reduce lamb birth weight (Warrington, et

al. 1988; Gao, et al. 2007). Brink (1990) suggested that the pregnant ewe that goes

through feed restriction during early pregnancy is able to compensate live weight and

body condition score during the later stages of pregnancy. Unfortunately, nutritional

restriction during pregnancy also has trans-generational consequences, compromising

the lifetime reproductive performance of the progeny (Gunn, et al. 1995; Rae, et al.

2002).

Ruminants can partially or completely compensate for an earlier period of slow

growth or body weight loss due to a low nutritional plane through increased feed intake

(reviewed by Hornick, et al. 2000; Daniel, et al. 2007). The magnitude and nature of

compensatory growth is influenced by factors such as severity of feed restriction, diet

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type during and after nutrient restriction, duration of nutrient restriction, breed and age

of the animal (Hornick, et al. 1998).

Mating ewe lambs

Mating ewe lambs at, say, 9 months of age rather than the traditional 18 months, is a

practice that has not been widely adopted, but recent studies indicate that the practice

can be successful. Resistance is due to perceptions of cost, poor reproductive

performance and a compromised performance in adult life. Quality of the ovum

(McMillan and McDonald 1985), ovulation rate (Davies and Beck, 1993; Beck, et al.

1996), pregnancy rate (Donald, et al. 1968; Annett and Carson, 2006; Quirke, 1981),

litter sizes at birth (Forrest and Bichard, 1974; Muñoz, et al. 2009) and lamb survival

(Morel, et al. 2010) have all been reported to be lower for ewe lambs than for older

ewes. Other potential disadvantages are: increase in farm costs, more labour required,

poor and variable reproductive performance, an increase in feed demand, greater live

weight and body condition score targets at a young age, decreased survival of newborn

progeny, tendency for progeny to be lighter at birth and weaning, and a greater rate of

deaths of the young ewes (reviewed by Kenyon, et al. 2013).

On the other hand, mating ewes as lambs enables an increase in the number of

lambs on the ground, and reduces the generation interval thus allowing the producer to

exploit potential advantages in sheep breeding and selection programs. Ewe lambs

need to weigh more than 40 kg at start of mating and, if they can rear lamb(s)

successfully to weaning, economic analyses suggest that the strategy will be profitable

if the lamb price is greater than $4/kg carcass weight and more than 60 lambs are

marked per 100 ewes mated (Coop, 1962; Young, et al. 2013). In addition, if the

females are well fed and their body condition is maintained, there is the potential to

increase lifetime reproductive performance (Schoeman, et al. 1995; Kenyon, et al.

2011). Finally, by gaining an extra year of production, successful early mating can lead

to a major reduction (theoretically 16% in a 6-year production life) in methane

emissions intensity in the production system.

Given these potential constraints and risks, producers need specific guidelines

for the reproductive management of young animals so they can maximize the chances

of success. For example, for young Merino ewes under Australian conditions,

producers need to know the optimum combination of live weight, liveweight change,

body condition and age for achieving puberty and maximising fertility.

Growth

Growth can be simply defined as an increase in body mass over time, but it must be

differentiated from development, in which the increase in mass is accompanied by a

11

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change in body composition. Body composition is the term used to describe the

percentages of fat, muscle and bone. For the purpose of brevity in this review, we will

exclude bone from the discussion.

The increase of body mass occurs during two specific stages, prenatal

(hyperplasia) and postnatal (hypertrophy), and involves regulated processes of growth

and differentiation that are modulated by a wide variety of factors. During the prenatal

stage, the mass of muscle and fat increases by cellular replication (hyperplasia) to

increase the number of cells; then, during the postnatal stage, those cells increase in

size (hypertrophy). As the animal matures, muscle cells lose their ability to replicate

and grow, but fat cells retain those capabilities and so continue to increase in number

and size (Berg and Butterfield, 1968; reviewed by Robelin, 1986; Azain, 2004). Growth

accelerates during the pre-pubertal stage then decelerates after puberty (Owens, et al.

1993; Robelin, 1986). The rate of change in body composition depends on internal and

external factors such as nutrition, age, gender, mature body size, genetics, and health

(Berg and Butterfield, 1968; Owens, et al. 1993; Nattrass, et al. 2006; Greenwood, et

al. 2006a and 2006b).

Adipose tissue

Adipose tissue is the body’s largest energy reservoir and is essential for regulating and

coordinating energetic and metabolic homeostasis (reviewed by Galic, et al. 2010). As

an energy storage site, adipose tissue enables the organism to adapt to a range of

different metabolic challenges (starvation, stress, periods of excess energy) and it has

also been implicated in endocrinological function through, primarily, the secretion of

leptin, a hormone that is involved in the modulation of puberty and fertility in a variety of

species (reviewed by Foster and Nagatani 1999; Casanueva and Dieguez 1999;

Frühbeck, et al. 2001; Smith, et al. 2002). The discovery of leptin corroborated the

hypothesis of Frisch (1984) that puberty and fertility are linked specifically to the

amount of fat stores.

Adipose tissue growth (adipogenesis) involves the proliferation, differentiation

and conversion of pre-adipocytes into adipocytes (reviewed by Hausman, et al. 2001;

Kokta, et al. 2004; Novakofski 2004; Fernyhough, et al. 2007). This process begins

during embryonic and fetal development (Hausman and Richardson 2004) and

continues during the postnatal stage (Azain, 2004), although the increase in fat mass in

this stage is mainly due to hypertrophy of adipocytes, a process that is prolonged by

over-nutrition (Lemonnier 1972; Faust, et al. 1978; Klyde and Hirsch 1979). Excess

energy is stored in adipocytes, the primary storage site (Azain, 2004; Nafikov and Beitz

2007), first in the abdominal cavity, then intermuscular spaces, subcutaneous regions

and, finally, intramuscular spaces (Vernon, et al. 1981; Pethick, et al. 2005). Variation

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in the ability to synthesize and store lipids in fatty tissues is closely linked to sex and

genotype and is reflected to the differences in total body fat. Moreover, the rate of

adipose tissue accumulation increases with age in meat animals (Owens, et al. 1993).

The interaction between genotype and nutrition was initially observed by Robelin

(1986) who concluded that fat deposition is more pronounced in the early maturing

animals due to the ability to store more excess of energy.

Adipose tissue is mainly classified as white, the main type, or brown (Cinti

2005). White adipose tissue is distributed throughout the body in subcutaneous regions

and plays a major role in energy storage and in the regulation of activities as diverse as

insulin sensitivity, lipid metabolism, and satiety (Trayhurn and Beattie 2001; Galic, et al.

2010), with flow-on consequences for many other tissues and processes, including

hypothalamus, pancreas, liver, skeletal muscle, kidneys and the immune system

(Frühbeck, et al. 2001). White adipose tissue accumulates rapidly in response to

nutrient availability (Kirtland and Harris 1980). It begins to develop in late gestation

(Vernon 1980; Tang, et al. 2008), increases in mass through puberty and is relatively

steady in maturity (Hirsch and Han 1969).

Brown adipose tissue is located in specific regions and mainly participates in

thermogenesis (reviewed by Rothwell and Stock 1979; Himms-Hagen, 1990; Cannon

and Nedergaard 2004). During late gestation, brown adipose tissue expands primarily

to maintain body temperature upon birth (Frontini and Cinti 2010).

Hormonal links between adipose tissue and reproduction are primarily mediated

by leptin (Fig. 3). Leptin secretion is strongly influenced by the immediate and long-

term nutritional status of the animal (Chilliard, et al. 2005) and its major role is

activation of the central satiety networks that suppress appetite (Zhang, et al. 1994). It

provides information to the hypothalamus about the quantity of energy stored in the

adipose tissue and, in response, the neural networks responsible for energy

homeostasis make appropriate changes in energy intake or expenditure to maintain

homeostasis. Leptin concentrations help to predict growth or carcass composition and

determine the body fat content of lambs (Altmann, et al. 2006), beef cattle (Geary, et al.

2003) and pigs (Berg, et al. 2003). Leptin, therefore, is involved in the direct regulation

of adipose tissue metabolism by both inhibiting lipogenesis (Bai, et al. 1996) and

stimulating lipolysis (Frühbeck, et al. 1997).

During growth and development, leptin also provides a signal to the brain

indicating whether metabolic status is adequate for the initiation of reproduction (Fig.

3). It acts directly on hypothalamic sites to enhance the secretion of GnRH and

gonadotrophins (reviewed by Nagatani, et al. 1998; Casanueva and Dieguez 1999;

Clarke and Henry 1999; Chilliard, et al. 2001). During the onset of puberty, leptin

concentration modulates the activity of kisspeptin (Backholer, et al. 2010), a

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neuropeptide that exerts fundamental control over the pulsatile secretion of GnRH

(Smith, et al. 2006; Navarro, et al. 2007; Smith and Clarke 2007). Several studies have

therefore shown that leptin mediates the effects of body weight and adiposity in

regulating the onset of puberty (Ahima, et al. 1997; Cheung, et al. 1997; Foster and

Nagatani 1999). For example, in immature rats, puberty can be induced by a re-feeding

stimulus for 72 h (Messer and I'Anson 2000) and by administration of recombinant

leptin (Cheung, et al. 1997; Almog, et al. 2001). Likewise, in female mice, leptin

treatment decreased food intake but advanced the onset of puberty (Ahima, et al.

1997; Chehab, et al. 1997) and re-activated the reproductive endocrine system of

males and females in the ob/ob genotype (Barash, et al. 1996). In Merino ewe lambs,

an intra-cerebroventricular infusion of leptin resulted in re-activation of the GnRH-LH

axis after it had been suppressed by fasting (Wójcik-Gladysz, et al. 2009).

Muscle tissue development

Myogenesis, the formation of muscle cells, takes place in two waves during prenatal

development. The first wave is during the initial stage of myoblast fusion and results in

fibres that are primary myoblasts or myofibres (Rehfeldt, et al. 2000). In the second

wave of myoblast proliferation, the primary myofibres serve as a template on which the

secondary muscle fibers develop (Stockdale 1997; Rehfeldt, et al. 2000). In the sheep,

myogenesis takes about 115 d (Greenwood, et al. 1999). The first wave of primary

myofibres occurs in the period leading up to 32-38 d of gestation and the second wave

from 38-62 d (Wilson, et al. 1992).

The number of primary muscle fibres produced during the hyperplasia phase

seems to be an important factor for muscle mass determination and it is regulated

mainly through genetic processes (Dwyer, et al. 1993 and 1994). Secondary muscle

development can be influenced by nutrition and by the addition of growth factors,

particularly IGF-I and GH (Rehfeldt, et al. 1993; Dwyer, et al. 1994; Greenwood, et al.

1999 and 2000). Fetal size (Greenwood, et al. 1999) and litter size (single vs twins)

(McCoard, et al. 2000) have no significant influence on muscle fibre number but, during

a seasonal restraint, the fibre number is differentially affected by season and fetal

number (McCoard, et al. 1997).

Hypertrophy is also markedly affected by the endocrine system, particularly

those that control the uptake and intracellular metabolism of nutrients. Among the

hormones implicated are growth hormone (GH), insulin, insulin-like growth factors

(IGFs), thyroid hormone (TH), transforming growth factor-ß (TGF-ß), glucocorticoids

(GC), insulin and leptin (Etherton 1982; Brameld, et al. 1998; Dauncey, et al. 2004;

Glass 2005; Zeidan, et al. 2005; Velloso, et al. 2008).

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Promoters and regulators of muscle growth and development

Myostatin (GDF-8) is a growth and differentiating factor that belongs to the

transforming growth factor beta (TGF-ß) super-family and has been identified as an

important inhibitory regulator of muscle development (McPherron, et al. 1997). It is

mainly synthesized in skeletal muscle, although low levels have been detected in

adipose tissue (McPherron, et al. 1997). It appears to regulate both prenatal

myogenesis and postnatal hypertrophy. During myogenesis, it regulates muscle fibre

number by controlling myoblast proliferation and differentiation whereas, during

postnatal stages, it regulates the activation of satellite cells (Thomas, et al. 2000; Lee

and McPherron 2001; McCroskery, et al. 2003).

A mutation in the myostatin gene is responsible for “double muscling” in some

breeds of cattle, such as the Belgian blue (McPherron and Lee 1997; Grobet, et al.

1997) and for enhanced muscle development in sheep (Clop, et al. 2006). In cattle,

myostatin concentration has been reported to differ between sexes within the same

breed and among breeds (Grobet, et al. 1997; Kambadur, et al. 1997; McPherron and

Lee 1997; McMahon, et al. 2003). Blockade of the myostatin signal increases the rate

of muscle accumulation and meat tenderness (Ngapo, et al. 2002), but has a negative

effect on fat depots (Zimmers, et al. 2002), skin, bones and reproductive performance

(poor sexual behaviour, delayed sexual maturity in both sexes, reduced size of the

reproductive tract in females, calving difficulty, poor maternal performance; Arthur

1995).

A promoter of muscle growth and development, and natural regulator of

myostatin, is follistatin (Lee and McPherron 2001). It plays an important role in muscle

growth and development during fetal development and early neonatal life (McFarlane,

et al. 2002). In mice, follistatin overexpression increases muscle weight and mass (Lee

and McPherron 2001; Lee 2007; Gilson, et al. 2009), corroborating the earlier work of

Matzuk, et al. (1995) who had reported retarded growth, reduced muscle mass, bone

deficiencies, and early postnatal death in follistatin-deficient mice. Therefore we would

expect follistatin concentrations to be high during muscle growth and development and

higher in ewe lambs selected for muscle.

Adipose, muscle and reproduction – possible interactions and hormonal links

As outlined above, the link between adipose tissue and reproduction has been clearly

identified, and the adipose hormone, leptin, seems to be a major player. In contrast, the

position is still not clear for links between the muscle tissue and the reproductive axis.

One possibility is follistatin – follistatin had no effect on GnRH secretory pattern

(Padmanabhan, et al. 2002) and its major role is to inhibit FSH secretion (Ueno, et al.

1987). Follistatin is also produced by the ovary although not exclusively and within the

15

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ovary, follistatin is synthesized mainly by granulosa cells and helps in the mechanism

of follicular selection (Michel, et al. 1990; Nakatani et al.,1991; Xiao et al 1992). During

ovulation, it seems that the role of follistatin is to bind and control activin actions; activin

which is important in ovulation (Nakamura, et al. 1990; Newton, et al. 2002). However,

the importance of follistatin on the reproductive physiology was demonstrated in mice;

deletion of follistatin in adult granulosa cells was found to altered follicle and oocyte

maturation, leading to reduced fertility and termination of ovarian activity (Jorgez, et al.

2004). Moreover, a deletion of one isoform of follistatin in mice reduced litter size and

early cessation of reproduction (Kimura, et al. 2010). It seems likely that changes in the

circulating concentration of follistatin will influence reproductive function (Fig. 3; review:

Knight, 1996).

An alternative is leptin from muscle – it appears to mediate the interactions

between muscle and fat cells that are important for adipogenesis, myogenesis and

lipogenesis (Boone, et al. 2000; Frühbeck, et al. 2001); It is expressed in muscle

(Wang, et al. 1998), where it induces and modulates hypertrophy (Zeidan, et al. 2005)

and exerts direct effects on glycogen and protein synthesis (Kamohara, et al. 1997; Liu,

et al. 1997; Carbó, et al. 2000), fatty acid oxidation (Muoio, et al. 1997) and basal and

glucose-induced insulin secretion (Emilsson, et al. 1997). Thus, follistatin and leptin

from muscle, as well as leptin from adipose tissue, could be involved in the control of

reproduction.

The genetics of body composition

Clearly, growth and body composition are complex traits that are controlled by different

internal factors that interact with each other as well as with the environment. In recent

times, these interactions have become important for the Australian sheep industry

because a strong interest has developed in genetic selection for fast growing animals

that accumulate more muscle than fat. Almost coincidentally, the selection for these

traits should permit reproduction at a younger age because the onset of puberty

depends on attainment of sufficient body mass and fertility and fecundity are highest in

females that are heavier at the start of the mating period. On the other hand, the

emphasis on accumulation of muscle rather than fat leads to a conundrum:

theoretically, the initiation of puberty depends on the adipose-derived hormone, leptin,

and its effects on the brain centres that control GnRH secretion. We would therefore

expect that animals selected for more accumulation of muscle rather than fat will

produce less leptin, thus delaying the onset of puberty. Evidence for an analagous

stimulatory role for muscle on reproduction, perhaps mediated by follistatin, is scanty

but it nevertheless suggests that animals selected for more accumulation of muscle

might experience an earlier onset of puberty and reproductive success. Support for this

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hypothesis comes from studies with adult ewes with higher values for yearling muscle

(YEMD) showing that fecundity increased (Ferguson et al., 2007).

Australian Sheep Breeding Values (ASBVs)

The performance of an animal is due to a combination of its genes and the

environment in which it is raised (eg, nutrition, birth type, rear type). To provide a single

language for genetic performance, and to improve the quality, scope and utilization of

sheep genetics in Australia, ASBVs were created by industry research organisations

and are delivered as LAMBPLAN™ for terminal and maternal breeds and

MERINOSELECT™ for Merino and Merino-based breeds. Both LAMBPLAN™ and

MERINOSELECT™ collate and analyse performance values, pedigree information and

relevant environmental and management information from participating breeders. The

outcome delivers ASBVs for a range of traits for growth, carcase, wool, reproduction

and parasite resistance, and it can be measured at four different stages: post-weaning

(7 to 10 months of age), yearling (10 to 13 months of age), hogget (13 to 18 months of

age), and adult (more than 18 months of age) (Brown, et al. 2007).

Genetic selection using sires with high estimated breeding values (EBVs) has

resulted in genotypes that vary in economically important traits such as weight (WT),

fat (FAT), muscle (EMD), fleece quality (clean fleece, CFW; fibre diameter, FD; staple

strength, SS; staple length, SL) number of lambs born (NLB), number of lambs weaned

(NLW), scrotal circumference (SC), and worm egg count (WEC).

Carcass and reproduction traits have recently become a focus of interest in the

Australian sheep industry because of sustained high prices for meat and low prices for

wool. Most emphasis has been placed on selection for growth, depth of ewe muscle

(EMD) and fat (FAT). Clearly, selection based on these traits is likely to change growth

response, carcass composition, carcass lean content and dressing percentage

(Hegarty, et al. 2006; Hopkins, et al. 2007; Gardner, et al. 2010). With the alteration in

body composition, we might expect that, in animals with higher values of ASBV for

rapid growth or improving accumulation of fat and muscle, there would be positive

effects on reproductive outcomes. However, at present, there is little information about

the impact this selection strategy may have on reproductive traits.

Therefore, for the present thesis, ASBVs will be a major tool for testing

hypotheses relating growth and body composition to puberty and reproductive

performance in young Merino ewes, and for testing possible roles for leptin and

follistatin as mediators of those relationships.

Conclusion

There is a wealth of literature demonstrating the importance of metabolic status in the

regulation of sexual maturation and reproductive performance, presumably due to the 17

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effects of physiological signals from metabolic tissues on the reproductive axis. The

role of adipose tissue and leptin has been studied extensively, and the action of leptin

on brain centres as a trigger for puberty has strong experimental support. On the other

hand, links between muscle and reproduction, especially in the determination of

puberty, are largely unexplored. Follistatin is one possibility – it plays an important role

in muscle growth and development during fetal and neonatal life and appears to be a

regulator of pituitary and ovarian acitivity, but not GnRH secretion. Overall, the situation

for follistatin is not clear, but the possibility that it will influence reproductive function is

worth testing.

Therefore, based on the literature, we expect that, in Merino ewe lambs, factors

that affect the rate of growth and accumulation of muscle and fat will influence the

concentration of follistatin and leptin, and thus the age and live weight at puberty, as

well as reproductive success.

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Chapter 1

Selection for superior growth advances the onset of puberty and increases reproductive performance in ewe lambs

C. A. Rosales Nieto1,2,3, M. B. Ferguson1,2,4, C. A. Macleay2, J. R. Briegel2, G. B. Martin3 and A.

N. Thompson1,2,4

1CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW, 2351,

Australia; 2Department of Agriculture and Food of Western Australia, 3 Baron Hay Court,

South Perth, Western Australia, 6151, Australia; 3Institute of Agriculture, University of Western

Australia, Crawley, Western Australia, 6009, Australia; 4School of Veterinary and Biomedical

Sciences, Murdoch University, 90 South Street, Murdoch, Western Australia 6150, Australia

Published in animal 2013; 7 990-997.

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Abstract The reproductive efficiency of the entire sheep flock could be improved if ewe lambs go

through puberty early and produce their first lamb at one year of age. The onset of

puberty is linked to the attainment of critical body mass so we tested whether it would

be influenced by genetic selection for growth rate or for rate of accumulation of muscle

or fat. We studied 136 Merino ewe lambs with phenotypic values for depth of eye

muscle (EMD) and fat (FAT) and Australian Sheep Breeding Values at post–weaning

age (200 days) for live weight (PWT), eye muscle depth (PEMD) and fat depth (PFAT).

First oestrus was detected with testosterone-treated wethers and then entire rams as

the ewes progressed from 6–10 months of age. Blood concentrations of leptin and IGF-

I were measured to test whether they were related to production traits and reproductive

performance (puberty, fertility, reproductive rate). Overall, 97% of the lambs reached

first oestrus at average weight 39.4 ± 0.4 kg (mean ± SEM) and age 219 days (range

163-301). Age at first oestrus decreased with increases in values for PWT (P<0.001)

and concentrations of IGF-I (P<0.05) and leptin (P<0.01). The proportion of ewe lambs

that achieved puberty was positively related with increased in values for EMD (P <

0.01), FAT (P<0.05) or PWT (P<0.01), and 75% of the ewe lambs were pregnant at

average weight 44.7 ± 0.5 kg and age 263 days (range 219-307). Ewe lambs that were

heavier at the start of mating were more fertile (P<0.001) and had a higher

reproductive rate (P<0.001). Fertility and reproductive rate were positively correlated

with increases in values for EMD (P<0.01), FAT (P<0.05), PWT (P<0.01) and leptin

concentration (P<0.01). Fertility, but not reproductive rate, increased as values for

PFAT increased (P<0.05). Leptin concentration increased with increases in values for

EMD (P<0.001), FAT (P<0.001), PWT (P<0.001), PEMD (P<0.05) and PFAT (P<0.05).

Many of these relationships became non-significant after PWT or live weight were

added to the statistical model. We conclude that selection for genetic potential for

growth can accelerate the onset of puberty and increase fertility and reproductive rate

of Merino ewe lambs. The metabolic hormones, IGF-I and leptin, might act as a

physiological link between the growing tissues and the reproductive axis.

Keywords: ewe lambs, phenotypic selection, ASBV, reproductive efficiency

Implications Genetic selection can improve the rates of growth and muscle development in sheep,

and should also permit reproduction at a younger age because the onset of puberty

depends on attainment of sufficient body mass. Our data support this hypothesis,

suggesting that phenotypic and genetic selection for growth or muscling will improve

the reproductive performance of Merino ewes mated to lamb at one year of age. These 20

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findings will inform bio-economic models and promote genetic selection strategies with

a view to improving profitability of sheep production systems by achieving

improvements in reproductive efficiency. The data also suggest a physiological link

between muscle and the reproductive system of female sheep, a possible new

direction in reproductive biology that needs further exploration.

Introduction International demand for lamb and the need to reduce emissions intensity are

increasing the emphasis on reproductive efficiency sheep flocks and renewing attention

on the breeding of young ewes in their first year of life (Martin et al., 2009; Ferguson et

al., 2011). Puberty, defined as the first spontaneous ovulation (reviewed by Foster et

al., 1985), is the result of dynamic interactions among several genetic and

environmental factors (reviewed by Dýrmundsson, 1981) and is generally reached in

ewe lambs when they attain 50 to 70% of their expected mature body mass (Hafez,

1952). If growth during early life is restricted, young ewes will remain pre-pubertal until

the required proportion of mature body mass is reached (Foster et al., 1985) so rapidly-

growing lambs achieve puberty earlier than slower-growing lambs (Boulanouar et al.,

1995). This relationship between growth rate and puberty explains the earlier puberty in

ewes raised as singles compared to those raised as twins across a range of breeds

(Southam et al., 1971). We would thus expect puberty to be advanced by phenotypic

and genetic selection for enhanced growth rate. This could be achieved through

selection of ewes with high Australian Sheep Breeding Values (ASBVs) for post–

weaning weight (PWT). As live weight at mating is related to fertility and reproductive

rate in young ewes (McGuirk et al., 1968), we would also expect that, at first mating,

ewes with higher growth rates would be more fertile and have higher reproductive rates

than ewes with lower growth.

In addition to the relationships between growth rate and early fertility, selection

strategies that alter the body composition of sheep might also be related to the timing

of first oestrus, fertility, and reproductive rate. In Merino sheep there are positive

genetic correlations between reproduction and growth and between reproduction and

body content of muscle and fat (Huisman and Brown, 2009). The fertility and

reproductive rate of adult Merino ewes is also known to be greater for genotypes with

higher body content of muscle and fat (Ferguson et al., 2007; 2010). Furthermore,

phenotypic enhancement in muscle mass and fatness, as assessed through condition

score, are known to increase fertility and reproductive rate in ewes mated to lamb at

one or two years of age (Malau-Aduli et al., 2007). Overall, it appears that ewe lambs

that accumulate fat and muscle rapidly will achieve puberty earlier, be more fertile and 21

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have a higher reproductive rate when mated at 8 or 9 months, than their counterparts

with lower rates of fat and muscle accumulation. Moreover, growth, fatness and the

onset of puberty are all associated with circulating concentrations of IGF-I and leptin

(Roberts et al., 1990; Chilliard et al., 2005), so we would expect reproductive

development to be explained by the secretory patterns of these two metabolic

hormones.

We tested these hypotheses by studying the relationships among phenotypic

values and ASBVs for rates of growth and accumulation of fat and muscle, plasma

concentrations of IGF-I and leptin, the timing of puberty and outcomes for fertility and

reproductive rate in Merino ewe lambs.

Material and Methods This work was done in accordance with the Australian Code of Practice for the Care

and Use of Animals for Scientific Purposes (7th Edition, 2004) and was approved by

the Animal Ethics Committee of the Department of Agriculture and Food, Western

Australia.

Experimental location and animals

The Merino ewe lambs (n = 136; 94 singles and 42 twins) used in this study were born

on a commercial farm (‘Moojepin’) in August–September 2009 to dams that had been

sourced from two Western Australian ram breeding flocks (‘Merinotech WA’ and

‘Moojepin’) and mated to sires with a wide range in Australian Sheep Breeding Values

(ASBV) for growth, muscle and fat. The ASBV values produced by MERINOSELECT

are the result of collation and analysis of individual performance measures, pedigree

information and relevant environmental and management information of the animals

from participating breeders.

The ewe lambs were transported to Medina Research Station (32.2° S,

115.8° E) where the experiment was conducted from February to June 2010. Live

weight (LW) was recorded weekly and the data were used to generate the average

daily gain (ADG). The depths of the longissimus dorsi muscle and subcutaneous fat at

a point 45 mm from the midline over the twelfth rib were measured using ultrasound

when the ewe lambs were aged 164 (range 134 to 176) and 251 (range 221 to 263)

days. The ultrasound data were used to generate phenotypic values for eye muscle

depth (EMD; range 20 to 33 mm) and C–site fat depth (FAT; range 2 to 8 mm). They

data were also used to generate ASBVs at post–weaning age for weight (PWT; range 0

to 9 kg), depth of eye muscle (PEMD; range 0 to 2.6 mm) and fat (PFAT; range 0 to 1.2

22

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mm) by MERINOSELECT (Brown et al., 2007). The ewe lambs were shorn when they

were on average 236 days old.

Animal management and feeding

The ewe lambs were initially run in two management groups in two 20 m x 60 m pens

with ad libitum access to clean water and sheep pellets that were introduced over a 7–

day period. The pellets based on barley, wheat and lupin grains, cereal straw and hay,

canola meal, minerals and vitamins were formulated to provide 11.5 MJ of

metabolisable energy per kg dry matter, 15% protein and minerals to meet their daily

requirements for maximum growth (Macco Feeds, Australia). On February 24 (Day -

69), when the ewe lambs were 179 days old (range 149 to 191) and weighed 37 ± 0.4

kg, four Merino wethers (rams castrated before puberty) with harnesses (MatingMark®;

Hamilton, NZ) were introduced to detect the onset of oestrus. The wethers had

received a 2 mL subcutaneous injection of testosterone enanthate (75 mg/mL; Ropel®,

Jurox, NSW) one week before they were placed with the ewe lambs. Every 2 weeks,

the injections were repeated and the crayons on the harnesses were changed. Crayon

marks on the rumps of ewe lambs were recorded three times per week to estimate the

date of standing oestrus. Date of oestrus was deemed as the date the first crayon mark

was recorded and age at this point were deemed to be age at puberty. The closest LW

recorded to the first crayon mark was deemed to be LW at puberty.

The wethers were removed at the end of this “teasing period”, on May 4 (day 0),

when the ewe lambs were 249 (range 219 to 261) days old and weighed 41 ± 0.5 kg.

For the “mating period”, the ewe lambs had received a 1 mL intramuscular injection of

supplement of vitamins (Vit A 500,000 iu; Vit D3 75,000 iu, Vit E 50 iu/mL; Vet ADE®,

Auckland, New Zealand) and were allocated, on the basis of LW and sire, into 8

management groups of 15 and moved into 3 m x 7 m pens where they had ad libitum

access to clean water and the sheep pellets. An experienced single Merino ram was

introduced into each group of ewe lambs. The rams were removed on day 47 and the

ewes remained indoors. Pregnancy and number of fetuses were confirmed by

ultrasound scanning 60 days after the rams were removed. The data from the mating

period were used to generate values for fertility (percentage of pregnant ewes per 100

ewes mated) and reproductive rate (number of fetuses in utero per 100 ewes mated).

The date the ewes lambed was recorded and conception date was deemed to have

occurred 150 days previously.

Blood sampling and immunoassay

Ewe lambs were not fasted and blood was sampled by jugular venipuncture on 5

occasions, when the ewe lambs were on average 199, 227, 248, 269 and 285 days old.

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Blood was collected into heparinised tubes, placed immediately on ice, and later

centrifuged at 2000 g for 20 min so plasma could be harvested and stored at - 20 ºC

until hormone analysis.

Plasma leptin concentrations were determined by radioimmunoassay in

duplicate 100 μL aliquots as described by Blache et al. (2000). The limit of detection

was 0.06 ng/mL and the intra-assay coefficient of variation was 7.3% at 0.73 ng/mL,

4.4% at 0.84 ng/mL, and 2.4% at 1.61 ng/mL. Plasma concentrations of IGF-I were

measured in duplicate samples using the RIA described by Gluckman et al. (1983).

Interference by binding proteins was minimized by acid–ethanol cryoprecipitation, as

validated for ruminants by Breier et al. (1991). The limit of detection for the assay was

0.05 ng/mL and the intra-assay coefficient of variation was 7% at 0.29 ng/mL and 5.1

% at 2.9 ng/mL.

Statistical analysis

Statistical analysis was aided by SAS version 9.3 (SAS, 2010). Changes in live weight

were analyzed using the linear mixed model procedures allowing repetitive measures

(PROC MIXED), with dam source, dam age (years), birth type and age at start of

mating as fixed effects. FAT, EMD, PWT, PEMD or PFAT were each independently

tested as covariates and dam age was used as a random effect.

The relationships among live weight, PWT, PEMD, PFAT, EMD and FAT were

computed using PROC GLM with the MANOVA option, allowing removal of major fixed

effects. Fixed effects included in the model were dam source, birth type and age at the

day of the muscle and fat scan.

Average daily gain (ADG) during the ‘Teasing’ and ‘Mating’ periods was

determined for each lamb using a cubic smoothing spline approach with transformation

regression model procedures, a method that is appropriate when the response is

nonlinear (TRANSREG). The ADG data were then analyzed using the linear mixed

model procedures (PROC MIXED). Fixed effects in the model were dam source, dam

age (years), birth type and age at start of the ‘Teasing’ or ‘Mating’ periods. FAT, EMD,

PWT, PEMD or PFAT were each independently tested as covariates, and dam age

was used as a random effect.

Age and live weight at first oestrus were also analysed using mixed models

(PROC MIXED) and included dam source, dam age (years), birth type and age as fixed

effects. FAT, EMD, PWT PEMD, PFAT, or concentration of leptin or IGF-I, were each

independently tested as covariates. Dam age was used as a random effect.

Data for puberty (marked or not) and fertility (pregnant or not) were analyzed

using the generalized linear mixed model procedures with a binomial distribution and

logit link function (PROC GLIMMIX). Fixed effects were dam source, dam age, birth

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type, age and live weight at the start of the period (teasing for puberty; mating for

fertility). Average daily gain (split into teasing for puberty and mating for fertility), FAT,

EMD, PWT, PEMD, PFAT, and leptin or IGF-I concentration, were each independently

tested as covariates. Dam age was used as a random effect. Reproductive rate (dry or

pregnant with single or twins) data was analyzed using the generalized linear mixed

model procedures with a multinomial distribution and logit link function (PROC

GLIMMIX). The same fixed effects, covariates and random effects were used as for the

analysis of fertility.

Data for blood concentrations of leptin and IGF-I were analysed using mixed

models (PROC MIXED) allowing for repeated-measures and included, as fixed effects,

dam source, dam age (years), birth type and age and live weight at start of teasing.

FAT, EMD, PWT, PEMD or PFAT were each independently tested as covariates.

Identification number of the ewe lamb within management group and date of sampling

were used as random effects. Mean metabolite concentrations were analyzed using

analysis of variance (repeated measures), where Factor A was hormone concentration

and Factor B was date at sampling (PROC ANOVA).

All 2-way interactions among the fixed effects were included in each model and

non-significant (P > 0.05) interactions were removed from the final model. The data for

puberty, fertility and reproductive rate are presented as logit values and back-

transformed percentages.

Mature LW was considered reached when ewes were more than two years old.

Therefore, to estimate it, individual records from LW and body condition score (BCS)

from the ewe lambs from birth to up to two years of age were used. Mature LW was

predicted at BCS 3 based on these records using linear regression of weight and BCS.

Results

Ewe live weight (LW)

From day –138 to day 55, mean (± SEM) LW increased from 24.2 ± 0.3 to 52.6 ± 0.5

kg (Figure 1). The ADG of the ewe lambs was 90 ± 2.5 g/d during the “teasing” period

and 214 ± 5.3 g/d during the “mating” period. Live weight during the experiment (both

periods) was positively related with increases in values for phenotypic traits (EMD or

FAT; P < 0.001) and ASBVs (PEMD or PFAT; P < 0.001). In general, live weight

increased by 1.4 kg as EMD increased 1 mm, by 3 kg as FAT increased by 1 mm, by

2.2 kg as PEMD increased 1 mm or by 4.4 as PFAT increased 1 mm. The ADG during

the “teasing” period was positively correlated with increases in values for EMD (P <

0.001) and FAT (P < 0.001). The ADG increased 4 g/d as EMD increased 1 mm or by

13 g/d as FAT increased by 1mm. PEMD or PFAT had no effect (P > 0.05) on the ADG

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during the “teasing” period. None of the variables had an effect on the ADG during the

“mating” period.

The correlations among LW, PWT, EMD, PEMD, FAT and PFAT are shown in

Table 1. Strong relationships were observed between PEMD and PFAT, EMD and FAT

and LW and EMD, whereas other relationships were relatively weak.

Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,

PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs.

Variable LW PWT EMD PEMD FAT PFAT

LW 1 0.68 0.64 0.28 0.56 0.29

PWT 0.68 1 0.53 0.40 0.39 0.34

EMD 0.64 0.53 1 0.41 0.59 0.34

PEMD 0.28 0.40 0.41 1 0.22 0.76

FAT 0.56 0.39 0.59 0.22 1 0.26

PFAT 0.29 0.34 0.34 0.76 0.26 1

Figure 1 Average live weight (± SEM) of single-born (●) or twin-born (○) Merino ewe lambs fed

ad libitum high quality pellet (11.5 MJ metabolizable energy per kg dry matter and 15% protein)

during the experiment. Day 0 is the day when Merino entire rams were introduced.

Live weight and age at puberty

Of the 136 lambs in the flock, 132 (97%) displayed oestrus during the “teasing” or

“mating” periods. The average weight at first oestrus was 39.4 ± 0.5 kg (range 26.9 to

55.1 kg) and the average age at first oestrus was 219 ± 3 days (range 163 to 301

-80 -60 -40 -20 0 20 40 6030

35

40

45

50

55

60 SingleTwin

Teasing period Mating period

Liveweight (Kg)

Days of the experiment

26

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days). The proportion of ewe lambs that attained puberty was influenced by both their

age (P < 0.05) and their LW (P < 0.01) at the beginning of the “teasing” period. After

adjustment for effects of LW, there were no effects of ewe source, dam age, birth type

or teasing group on the proportion of ewe lambs that attained puberty (Table 2). The

ADG during the “teasing” period had no effect on the proportion of ewes that reached

puberty. The proportion of ewe lambs that attained puberty was positively related with

increases in values for PWT (P < 0.01; Figure 2), EMD (P < 0.01) and FAT (P < 0.05).

The ASBVs for PEMD or PFAT had no effect on the likelihood of a ewe reaching

puberty (Table 3).

Table 2: Effect of classification variables [Birth type (BT), rear type (RT), ewe source and dam

age] on reproductive performance [age or live weight at first oestrus, puberty, fertility and

reproductive rate (Rep Rate)] and on metabolic hormone concentration (IGF-I or leptin) in

Merino ewe lambs.

Variable Type Age at

1st oestrus LW at

1st oestrus Puberty Fertility

Rep Rate

IGF-I Leptin

Days Kg (%) (%) (%) ng/mL ng/mL

BT NS NS NS NS ** NS NS

Single 218 39.8 96 81 64.7 1.59

Twin 222 38.5 100 62 61.6 1.51

RT NS NS NS NS NS NS NS

Single 214 39.5 98 76 64.6 1.55

Twin 221 39.2 95 72 61.6 1.60

Ewe source *** * NS NS NS NS * Moojepin 209 38.7 97 77 63.9 1.60

MerinoTech 234 40.8 97 72 63.6 1.51

Dam age (years) NS NS NS NS NS NS NS

1.5 225 39.1 97 77 61.7 1.56

2.5 210 40.2 100 74 70.1 1.62

3.5 219 40.9 100 89 62.6 1.65

4.5 217 38.8 95 74 62.6 1.50

P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05

The average LW and age at the beginning of the “teasing” period was 36.8 ±

0.4 kg and 179 ± 1 days (range 149 to 191 days). Live weight and age at first oestrus

differed with ewe source (P < 0.05; P < 0.001), but the other variables tested had no

effect on either LW or age at first oestrus (P > 0.05). There was no relationship

between the ADG and age at first oestrus (P > 0.05; Table 3). On average, of the

lambs that achieved puberty, twin–born lambs were 1.3 kg lighter and 4 days older

than single lambs at their first oestrus (38.5 vs 39.8 kg and 222 vs 218 days).

27

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Live weight at first oestrus was estimated to be 62% of mature body weight.

The average mature body LW was 63.7 ± 0.7 kg (range 49 – 86). Live weight at first

oestrus was positively correlated with increases in values for PFAT (P < 0.01), PWT (P

< 0.001) or EMD (P < 0.001) and it increased by 0.3 kg for each 1 mm of PFAT, by 2.3

kg for each 1 kg increase in PWT and by 1.2 kg for each 1 mm of EMD. Neither PEMD

nor FAT had effect on LW at first oestrus (Table 3).

Ewe lambs with higher values for PEMD (P < 0.05) or PWT (P < 0.001; Figure

3) were younger at first oestrus than ewe lambs with lower values for those traits. Age

at first oestrus decreased by 1 day as PEMD increased 1 mm or by 7 days as PWT

increased 1 kg. PFAT, EMD and FAT had no effect on age at first oestrus (Table 3).

After LW at scanning was included in the statistical model for EMD or FAT or

PWT for PEMD; the effect of EMD and FAT on puberty and PEMD on age at first

oestrus was no longer evident.

Table 3: Effect of phenotype [Eye muscle depth (EMD) and fatness (FAT)], ASBV for post-

weaning weight (PWT), post-weaning eye muscle depth (PEMD), or post-weaning fatness

(PFAT), or metabolic hormone concentration (IGF-I or leptin) on reproductive performance (age

or live weight at first oestrus, puberty, fertility and reproductive rate) or metabolic hormone

concentration (IGF-I or leptin) in Merino ewe lambs.

Variable Age at 1st oetrus

LW at 1st oestrus

Puberty Fertility Rep Rate

IGF-I Leptin

Days Kg (%) (%) (%) ng/mL ng/mL

Age (Teasers in) NA NS * NA NA NA NA

LW (Teasers in) NS NA ** NA NA NA NA

Age (Rams in) NA NA NA NS NS NS *

LW (Rams in) NA NA NA *** *** NS ***

ADG (Teasing) NS NA NS NA NA NA NA

ADG (Mating) NA NA NA NS NS NA NA

PWT *** *** ** ** ** NS ***

PEMD * NS NS NS NS NS *

PEMD (+PWT) NS NA NS NS NS NA NS

PFAT NS ** NS * NS NS *

PFAT (+PWT) NS NA NS NS NA NA NS

EMD NS *** ** ** ** NS ***

EMD (+LW@scan) NS NA NS NS NS NA *

FAT NS NS * * * NS ***

FAT (+LW@scan) NA NA NS NS NS NA ***

IGF * NS NS NS NS NA NA

IGF (+LW@sampling) NS NA NS NS NS NA NA

Leptin ** NS NS ** ** NA NA

Leptin (+LW@sampling) NS NA NS * * NA NA

P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05; NA No applicable 28

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Figure 2 Relationships between Australian Sheep Breeding Value for post–weaning weight

(PWT) and the proportion of Merino ewe lambs that achieved puberty by the end of mating

when their average age was 296 days. The dashed lines represent upper and lower 95%

confidence limits.

Figure 3 Relationships between Australian Sheep Breeding Value (ASBV) for post-weaning

weight (PWT) and age at first oestrus in Merino ewe lambs. Data from single and twin births

were all retained in the model and the relationship was pooled within the birth-type classes. All

lambs were fed ad libitum with high quality pellets from 6 to 10 months of age. The dashed lines

represent upper and lower 95% confidence limits.

70

80

90

100

0 1 2 3 4 5 6 7 8 9Post weaning weight (PWT) ASBV (Kg)

Puberty reached (%)

170180190200210220230240250

1 2 3 4 5 6 7 8 9Post weaning weight (PWT) ASBV (Kg)

Age at first oestrus (Days)

29

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Fertility and reproductive rate

A total of 102 out of 136 (75%) ewe lambs were pregnant. Fertility was positively

related to liveweight at start of mating. Ewe lambs that were heavier at the start of

mating were pregnant (P < 0.001; Figure 4). Of those that conceived, the average

weight and age at pregnancy was 44.7 ± 0.5 kg (range 35.3 to 59.2 kg) and 263 ± 2

days (range 219 to 307 days). The ADG during the “mating” period had no effect on

fertility. Fertility differed with birth type (P < 0.05) and mating sire (P < 0.05), but the

rest of the variables had no effect (Table 2). On average, twin–born lambs were 0.6 kg

lighter and 7 days older than single lambs at pregnancy (44.3 vs 44.9 kg and 268 vs

261 days).

The pregnancy rate was positively related with increases in values for PWT (P <

0.01; Figure 5), PFAT (P < 0.05), EMD (P < 0.01), FAT (P < 0.05), but these

relationships, except PWT, were explained by correlated changes in LW and

disappeared when LW or PWT was added to the model. The ASBV for PEMD had no

effect on fertility (Table 3).

Figure 4 Relationships between live weight at the start of mating and fertility of Merino ewe

lambs between 6 and 10 months of age. Data from single and twin births were all retained in the

model and the relationship was pooled within the birth-type classes. All lambs were fed ad

libitum with high quality pellets. The dashed lines represent upper and lower 95% confidence

limits. Of the ewe lambs that were pregnant, 84% were carrying a single lamb and

16% were carrying twins. Reproductive rate differed with birth type (P < 0.01).

Reproductive rate was positively related to LW at start of the mating period (P < 0.001).

On average each extra kg at start of mating was associated with extra 4.8 fetuses per

100 ewes (P < 0.001; Figure 6). Reproductive rate was positively correlated with

20

40

60

80

100

30 35 40 45 50 55

Fertility (%)

Liveweight (Kg)

30

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increases in values for PWT (P < 0.01), EMD (P < 0.01) and FAT (P < 0.05). But, the

effect of EMD or FAT on reproductive rate was no longer evident after LW was added

into the statistical analyses.

Figure 5 Relationships between Australian Sheep Breeding Value (ASBV) for post–weaning

weight (PWT) and the proportion of single- and twin-born Merino ewe lambs that conceived

between 7 and 10 months of age. All lambs were fed at libitum with high quality pellets. The

dashed lines represent upper and lower 95% confidence limits.

Figure 6 Relationships between live weight at start of mating and proportion of overall lambs of

Merino ewe lambs fed ad libitum high quality pellets (15% protein) between 6 and 10 months of

age. The dashed lines represent upper and lower 95% confidence limits.

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9

Single

Twin

Fertility (%)

Post weaning weight (PWT) ASBV (Kg)

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

180.0

35.0 40.0 45.0 50.0

Overall lambs per 100 ewes

Liveweight (Kg)

31

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Hormone profiles

As the experiment progressed, circulating concentrations of leptin increased from 1.31

± 0.02 to 1.78 ± 0.01 ng/mL (P < 0.001) and concentrations of IGF-I increased from

39.2 ± 1.3 to 85 ± 2.2 ng/mL (P < 0.001). Leptin concentrations differed with ewe

source (P < 0.05), birth type (P < 0.05) and date of sampling (P < 0.001).

Concentrations of IGF-I differed with date of sampling (P < 0.001), but the other

variables were not related to IGF-I values (Table 2).

The concentrations of IGF-I or leptin were negatively correlated with age at first

oestrus, so first oestrus was advanced as leptin (P < 0.01) and IGF-I (P < 0.05)

concentrations increased. Ewe lambs were younger at first oestrus by 0.2 days as IGF-

I concentration increased 1 ng/mL or by 28 days as leptin increased 1 ng/mL.

Concentrations of IGF-I and leptin were not related to LW at first oestrus or puberty

(Table 3). The concentration of leptin, but not IGF-I, was positively related to fertility

and to reproductive rate (P < 0.01; Table 3).

Ewe lambs with higher values for PWT (P < 0.001), PEMD (P < 0.05), PFAT (P

< 0.05), EMD (P < 0.001) and FAT (P < 0.001) had a greater leptin concentration than

ewe lambs with lower values. Leptin increased by 0.05 ng/mL as PWT increased 1 kg,

by 0.05 ng/mL as PEMD increased 1 mm, by 0.01 ng/mL as PFAT increased 1 mm, by

0.03 ng/mL as EMD increased 1 mm or by 0.09 ng/mL as FAT increased 1 mm. PEMD

was not associated with leptin concentration. Neither ASBV nor phenotypic values had

effect on IGF-I concentrations (Table 3). The effect of leptin on age at first oestrus and

ASBV was no longer evident once LW was added in the statistical analysis. However,

after adjustment for effects of LW, the effect of leptin concentration on fertility,

reproductive rate, EMD or FAT remained evident. Discussion The data support the hypothesis that age and LW at first oestrus, onset of puberty,

fertility and reproductive rate are all influenced by higher values for post-weaning

growth. These observations extend those of Hawker and Kennedy (1978) and Alkass

et al. (1994) who showed that ewes that grew faster reached puberty at a younger age.

In addition, our observations agree with those of Kenyon et al. (2010) who observed

that ewe lambs that attained heavier LW and higher condition score at mating were

more likely to get pregnant and improve the fecundity rate. Decades of research has

shown that we need to provide high quality nutrition to young ewes so they can reach

puberty in a timely manner (Cave et al., 2012) but the present study has also shown

that we can also achieve that aim whilst developing lean carcasses through genetic

selection. 32

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The fundamental relationship between body mass and the onset of puberty was

not challenged because the average LW of the lambs that reached puberty was about

62% of their mature LW and thus within the critical 50 to 70% range (Hafez, 1952). On

the other hand, there seems to be a critical LW around 45 kg at the start of mating,

reached earlier in faster growing ewe lambs, where fertility improves. We observed a

linear response between pregnancy rate and LW at the start of mating, over the range

30 to 45 kg, expanding upon previous observations in 18-month-old maiden ewes by

Kleemann and Walker (2005). However, once the 45 kg point had been exceeded, the

response became curvilinear. There was also positive linear effect of LW at start of

mating on reproductive rate, consistent with our observations on mature ewes

(Ferguson et al., 2011), with perhaps greater benefit of additional weight for ewe lambs

than for mature ewes. For ewe lambs, each extra kg was associated with 4.8 extra

fetuses per 100 ewes in contrast with 1.7-2.4 extra fetuses for mature ewes (Ferguson

et al., 2011). This increases the value of reaching the critical live weight at start of

mating for ewe lambs.

High ASBV values for growth can also improve fertility and reproductive rate

because ewe lambs with higher values for PWT achieve the critical percentage of

mature LW earlier and are more suitable for mating at younger ages, as reported

previously (McGuirk et al., 1968). This reflects the positive genetic correlation between

weaning weight and fertility (Barlow and Hodges, 1976). On the other hand, in the

present study, ADG during the “mating” period had no effect on fertility or reproductive

rate, perhaps because the pregnancy rates were already maximal with ADG at high

values (more than 200 g per day). Additionally, the ewes that conceived presumably

did so during the second or third cycle after their first oestrus, as observed by Hare and

Bryant (1985). The remainder of the ewes were detected in oestrus by either teasers or

rams but failed to conceive, perhaps reflecting low quality of ovum or a high incidence

of pre-natal mortality (Quirke, 1981; McMillan and McDonald, 1985). Despite this

problem, it is clear that genetic strategies that increase growth rate will not only

advance puberty but also result in greater fertility and reproductive rate in Merino ewe

lambs.

The data support the hypotheses that age and LW at first oestrus, puberty,

fertility and reproductive rate are all influenced by the rate of accumulation of muscle or

fat. The genetic correlations that we observed among live weight and depths of muscle

and fat were small but positive, whereas the phenotypic correlations observed among

live weight and depth of muscle and fat, also positive, were stronger. However, an

important aspect of the present study is the dissection of effects based on LW, a

passive endpoint, into effects that can be specifically attributed to major, physiologically

active body tissues. Thus, the relationship between onset of puberty and the rate of

33

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accumulation of muscle and fat is supported by the existence of endocrine factors from

both tissues that are thought to directly affect the brain processes that control the

initiation of puberty. We found that circulating concentrations of leptin and IGF-I

increased progressively as the experiment progressed and puberty approached,

consistent with previous reports (Roberts et al., 1990; Foster and Nagatani 1999),

suggesting that the two metabolic hormones inform the central nervous system of the

metabolic status of the body, perhaps specifically the accumulation of fat (leptin) and

muscle (IGF-I), and thus permit the triggering of puberty. This role for leptin has been

largely confirmed in ewe lambs, but the question still remains open for IGF-I or any

other endocrine factor associated with muscle.

After puberty, leptin (but not IGF-I) might also explain the relationships between

fertility and reproductive rate and the accumulation of muscle and fat in both young

ewes (present study) and mature Merino ewes (Ferguson et al., 2007; 2010). We found

that the concentration of leptin was associated with fertility and reproductive rate,

consistent with earlier evidence linking leptin to the regulation of fertility (reviewed by,

Smith et al., 2002). Furthermore, ewe lambs with higher phenotypic values for EMD or

FAT or ASBVs for PWT, PEMD or PFAT had greater leptin concentrations than ewe

lambs with lower values for those traits. Interestingly, when LW was added in the

statistical model for EMD and FAT, the effect of these traits on leptin concentration

remained. This might be expected, since animals selected for muscling tend to have

bigger muscles and be bigger and heavier and, since leptin is produced by adipose

tissue, changes in leptin concentration are driven by changes in LW (Blache et al.,

2000). It seems that muscle and fat accumulation modifies circulating leptin, exerting a

positive influence on the reproductive performance of Merino ewe lambs.

Conclusion Live weight at the start of mating is an important determinant of the reproductive

performance of ewe lambs, and the present study shows that we can address this

limitation in Merino sheep by using genetic strategies for increasing the rates of muscle

and fat accumulation and thus advancing puberty and increasing fertility and

reproductive rate. Our data also suggest that there is a physiological link between

muscle and the reproductive system of female sheep, a novel hypothesis that needs

further investigation. For the sheep industry, these findings should promote genetic

selection strategies that improve profitability by improving reproductive efficiency, and

also help establish modern systems of animal management that will reduce emissions

intensity (Martin et al., 2009).

34

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Acknowledgements The authors wish to thank to Andrew Kennedy and David and Hamish Thompson for

their assistance with collection of pedigree data and Margaret Blackberry for her help

with hormone analyses. Cesar Rosales Nieto was supported by CONACyT (the

Mexican National Council for Science and Technology), the Department of Agriculture

and Food of Western Australia, the Cooperative Research Centre for Sheep Industry

Innovation, and the UWA School of Animal Biology during his doctoral studies.

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Ferguson MB, Young JM, Kearney GA, Gardner GE, Robertson IRD and Thompson AN 2010.

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Huisman AE and Brown DJ 2009. Genetic parameters for bodyweight, wool, and disease

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Kenyon PR, Morris ST and West DM 2010. Proportion of rams and the condition of ewe lambs

at joining influences their breeding performance. Animal Production Science 50, 454-459.

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in the uteri of ewe lambs. Animal Reproduction Science 8, 235-240.

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Roberts CA, McCutcheon SN, Blair HT, Gluckman PD and Breier BH 1990. Developmental

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Chapter 2

Ewe lambs with higher breeding values for growth achieve higher reproductive performance when mated at age 8 months

C.A. Rosales Nietoa,b,c, M.B. Fergusona,b,d,1, C.A. Macleayb, J.R. Briegelb, D.A. Woodb,

G.B. Martinc, A.N. Thompsona,b,d,*

a CRC for Sheep Industry Innovation and the University of New England, Armidale, New South

Wales, 2351, Australia b Department of Agriculture and Food of Western Australia, 3 Baron Hay Court, South Perth,

Western Australia, 6151, Australia c UWA Institute of Agriculture, University of Western Australia, Crawley, Western Australia,

6009, Australia d School of Veterinary and Biomedical Sciences, Murdoch University, 90 South Street, Murdoch,

Western Australia 6150, Australia 1 Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch 8024,

New Zealand

Published: Theriogenology 2013; 80, 427-435

38

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Abstract We studied the relationships among growth, body composition and reproductive

performance in ewe lambs with known phenotypic values for depth of eye muscle

(EMD) and fat (FAT) and Australian Sheep Breeding Values for post-weaning live

weight (PWT) and depth of eye muscle (PEMD) and fat (PFAT). Vasectomized rams

were placed with 190 Merino ewe lambs, when they were, on average, 157 days old, to

detect first oestrus. The vasectomized rams were replaced with entire rams when the

ewe lambs were, on average, 226 days old. Lambs were weighed every week and

blood was sampled on four occasions for assay of ghrelin, leptin and ß-

hydroxybutyrate. Almost 90% of the lambs attained puberty during the experiment, at

an average live weight of 41.4 kg and average age of 197 days. Ewe lambs with higher

breeding values for EMD (P < 0.001), FAT (P < 0.01), PWT (P < 0.001), PEMD (P <

0.05) and PFAT (P < 0.05) were more likely to achieve puberty by 251 days of age.

Thirty-six percent of the lambs conceived and, at the estimated date of conception, the

average live weight was 46.9 ± 0.6 kg and average age was 273 days. Fertility,

fecundity and reproductive rate were positively related to PWT (P < 0.05) and thus live

weight at the start of mating (P < 0.001). Reproductive performance was not correlated

with blood concentrations of ghrelin, leptin and ß-hydroxybutyrate. Many ewe lambs

attained puberty, as detected by vasectomised rams, but then failed to become

pregnant after mating with entire rams. Nevertheless, we can conclude that, in ewe

lambs mated at 8 months of age, higher breeding values for accumulation of muscle

and fat, and therefore growth, are strongly positively correlated with reproductive

performance, although the effects of breeding values for growth and responses to live

weight are highly variable.

Keywords: ewe lambs, reproductive performance, phenotypic selection, Australian

sheep breeding value (ASBV)

39

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1. Introduction

Puberty in females is defined by the first ovulation because that is when

reproduction becomes possible. In ewe lambs, it is usually achieved when they attain

50-70% of their mature live weight, with age per se being a secondary factor [reviewed

by 1,2]. Because of the importance of live weight, environmental factors that affect the

rate of growth before and after weaning are important determinants of age at puberty

[reviewed by 3]. For example, ewe lambs born and raised as singles reach puberty at a

younger age than those born and raised as twins [4], because single-reared lambs

grow faster to weaning and can remain heavier until 12 months of age despite post-

weaning compensatory growth in twin-reared progeny [5]. As genetic potential for

growth also affects live weight, it is not surprising that Merino ewe lambs with higher

breeding values for growth are younger at puberty and achieve greater fertility and

reproductive rate when mated at 8-10 months of age [6,7].

In addition to the effect of live weight, we have demonstrated in adult ewes the

relationships of fertility and fecundity with the accumulation of muscle and fat [8,9], and

we have begun extending these studies to ewe lambs. We have shown that, under

animal house conditions, ewe lambs with higher breeding values for muscle and fat

attain puberty earlier and are more likely to conceive than ewe lambs with lower

breeding values [7]. Muscle and fat are genetically correlated with live weight [10] but,

in adult sheep, the relationships between muscle and fat on fertility and reproductive

rate were not related to live weight, in contrast with the situation in ewe lambs [7]. The

potential effect of genetically accelerated fat accumulation on reproduction is likely to

be reduced under conditions of good nutrition [9] and good nutritional conditions were

maintained throughout the previous study of ewe lambs [7]. This might have diminished

the impact of these metabolic tissues on their reproductive performance.

Sexual maturation and reproductive performance are closely linked to the

metabolic status of the animal, due to the effects of physiological signals from

metabolic tissues on the reproductive axis [11]. There are two primary candidate from

adipose tissue: i) leptin, a potent regulator of appetite, metabolism and reproductive

endocrinology [12]; and ii) β-hydroxybutyrate, an indicator of energy status when there

is a relatively high glucose demand [13]. From the gut, a likely candidate is ghrelin, a

hormone that acts as a signal of energy insufficiency and thus generally inhibits the

reproductive axis [14]. We therefore tested the hypotheses that phenotypic and

genotypic values for growth, and for accumulation of fat and muscle, will affect the

timing of puberty and the reproductive performance of ewe lambs mated under

extensive conditions, and that these relationships will be related to circulating

concentrations of leptin, ghrelin and β-hydroxybutyrate.

40

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2. Material and Methods This experiment was undertaken in accordance with the Australian Code of Practice for

the Care and Use of Animals for Scientific Purposes (7th Edition, 2004) and was

approved by the Animal Ethics Committee of the Department of Agriculture and Food,

Western Australia.

2.1. Experimental location and animals

The Merino ewe lambs (n = 190; 135 singles and 55 twins) used in this

experiment were born in June 2010 at the research farm of the University of Western

Australia, near Pingelly in Western Australia (32.2° S, 115.8° E). The dams of the ewe

lambs had been sourced from two ram breeding flocks in Western Australia and the

sires had a range in Australian Sheep Breeding Values for growth, muscle and fat

accumulation. The Australian Sheep Breeding Values produced by MERINOSELECT

are the result of collation and analysis of individual performance values, pedigree

information and relevant environmental and management information of the animals

from participating breeders. Data for birth date, birth weight, birth type and rear type to

weaning were collected for the ewe lambs and, on 1 November 2010, they were

transported to the Medina Research Station (32.2° S, 115.8° E) for the first stage of the

experiment. In late December, they returned to the university farm where they

remained until the end of the experiment (Fig. 1).

The ewe lambs were weighed weekly from the start of ‘teasing’ (the introduction

of vasectomized adult rams) on November 30 (Day -70; Day 0 was to the start of the

mating period with intact rams on February 8) to the end of the experiment (Day +98).

Live weight data were interpolated to estimate the live weight at puberty and the

estimated date of conception and to calculate the average daily gain (ADG). After Day

+ 98, the ewe lambs were weighed and condition scored every three months until two

years of age to predict mature live weight. Mature live weight was predicted as the

conceptus-free live weight at condition score 3 from a linear regression of the live

weight and condition score data for each animal.

The depth of the longissimus dorsi muscle and subcutaneous fat at a point 45

mm from the midline over the twelfth rib was measured using ultrasound when the

average age of the ewe lambs was 167 (range 146–186) and 218 (range 198–228)

days. For both measurements, the range in eye muscle depth (EMD) was 20–33 mm

and the range in C-site fat (FAT) was 2–8 mm. Using MERINOSELECT [15], the

ultrasound data were used to generate estimates of Australian Sheep Breeding Values

at post-weaning age for weight (PWT; range 0–9 kg), depth of eye muscle (PEMD;

range 0.0–2.6 mm) and depth of fat (PFAT; range 0.0–1.2 mm). 41

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2.2. Animal management and feeding

At the Medina research station, ewe lambs were allocated on the basis of live

weight among eight groups, each housed in a 6 x 14 m pen where they had ad libitum

access to water and to pellets that were introduced over a 7-day period. The pellets

were based on barley, wheat and lupin grains, cereal straw and hay, canola meal,

minerals and vitamins. They were formulated to provide 11.5 MJ of metabolisable

energy per kilogram of dry matter, 15% protein, and minerals and vitamins for

maximum growth. At the start of ‘teasing’, the ewe lambs were on average 157 days

old (range 136–176) and weighed 36.2 ± 0.3 kg (range 24.8–50.8). A vasectomized

Merino ram with a marking harness (MatingMark®; Hamilton, NZ) was introduced into

each pen to detect the onset of oestrus. On December 29 (Day - 41), the ewe lambs

and vasectomized rams were moved to the University farm (Fig. 1) where each of the

eight groups was allocated to a separate 30 x 120 m field.

The vasectomized rams were removed on February 8 (Day 0) and ewe lambs

were allocated on the basis of their live weight and prospective sire into 8 groups with

23–24 ewe lambs per group. An experienced ram with a marking harness was

introduced into each group to begin “Mating period 1” (Fig. 1) when the ewe lambs

were on average 226 days old (range 206–246) and weighed 42.4 ± 0.3 kg (range

24.3–56.4). The rams were removed after 24 days. One month later, after a pregnancy

scan indicated a low fertility rate, a second set of eight experienced rams were

introduced to all ewe lambs, thus beginning “Mating period 2” (Fig. 1). At this stage, the

ewe lambs were on average 285 days old (range 264–304) and weighed 48.4 ± 0.3 kg

(range 29.0–61.5). These rams were removed 21 days later. Throughout both mating

periods, the lambs had access to clean water and had ad libitum access to oaten hay

(9 MJ/kg and 9% protein) plus lupin grain (13.5 MJ/kg and 32% protein). It was

anticipated that the combination of supplement plus dry pasture would allow the lambs

to gain about 100 g/day. Crayon marks on the rumps were recorded three times per week at Medina and

once per week at the university farm to estimate the date of their first standing oestrus.

Crayon marks were scored at 1, 2 or 3, with score 1 being one narrow mark on the

middle or the edge of the rump and score 3 as being a single large mark covering the

rump. The date when the first score 2-3 crayon mark was recorded was deemed to be

the age at first oestrus and the closest live weight recorded to that date was deemed to

be live weight at first oestrus. Pregnancy rate and the number of fetuses per ewe were

measured at ultrasound scanning in mid-April and mid-June. The data from both

mating periods were combined and used to generate values for fertility (percentage of

pregnant ewes per 100 ewes mated), fecundity (percentage of pregnant ewes with twin

42

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fetuses) and reproductive rate (number of fetuses in utero per 100 ewes mated). The

date the ewes lambed was recorded and conception date was deemed to have

occurred 150 days previously. Fig. 1. A schema describing animal management during the experiment, with Day 0 designated

as the start of the first mating period.

2.3. Blood sampling and assay

Blood (5 ml) was sampled into heparinised tubes by jugular venipuncture when

the ewe lambs were on average 144 (Day - 83), 186 (Day - 39), 227 (Day 0) and 254

(Day + 28) days old. The samples were immediately placed on ice and then centrifuged

at 2000 g for 20 min so plasma could be harvested and stored at –20º C until hormone

analysis. Ghrelin was measured in duplicate 100 μL aliquots of plasma with a double-

antibody radioimmunoassay (RIA) method, as modified by Miller et al. [16]. The limit of

detection was 49 pg/mL and the intra-assay coefficient of variation was 6% at 94

pg/mL, 2.6% at 327 pg/mL, and 1% at 1550 pg/mL. Plasma leptin was measured in

duplicate 100 μL aliquots using the double antibody RIA described by Blache et al.

[17]. The limit of detection was 0.05 ng/mL and the intra-assay coefficient of variation

was 16% at 0.47 ng/mL, 3.3% at 1.10 ng/mL, and 3.6% at 1.79 ng/mL. Concentrations

of ß-hydroxybutyrate were determined on an automated analyser (AU400, Olympus,

Tokyo, Japan) using the RANDOX RANBUT reagents supplied by Perth Scientific.

2.4. Data analysis

Statistical analysis was aid by SAS version 9.3 [18]. Changes in live weight

were analyzed using the linear mixed model procedures allowing repetitive measures

(PROC MIXED), with dam source, dam age (years), birth type and age at start of

mating as fixed effects. FAT, EMD, PWT, PEMD or PFAT were each independently

tested as covariates and the identification number of ewe lamb within sire (father)

group was used as a random effect.

Ewe lambsborn

Transportto Medina

Teasing period

Return toUWA farm

UWA farm Mating period 1 Mating period 2

Feb 8–Mar 42011

7–29 Apr2011

-252 -99 -70 -41 0 5824 80Day of experiment

Jun2010

Nov2010

29 Dec2010

43

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The relationships among live weight, PWT, PEMD, PFAT, EMD and FAT were

computed using PROC GLM, with MANOVA option, allowing removal of major fixed

effects. Fixed effects included in the model were dam source, birth type and age at the

day of the muscle and fat scan.

Average daily gain (ADG) during the ‘Teasing’ and ‘Mating’ periods was

determined for each lamb using a cubic smoothing spline approach with transformation

regression model procedures, a method that is appropriate when the response is

nonlinear (TRANSREG). The ADG data were then analyzed using the linear mixed

model procedures (PROC MIXED). Fixed effects in the model were dam source, dam

age (years), birth type and age at start of the ‘Teasing’ or ‘Mating’ periods. FAT, EMD,

PWT, PEMD or PFAT were each independently tested as covariates, and the sire

(father) of the ewe lamb was used as a random effect.

Age and live weight at first oestrus were also analysed using mixed models

(PROC MIXED) and included dam source, dam age (years), birth type and age as fixed

effects. FAT, EMD, PWT PEMD, PFAT or concentration of leptin or ghrelin were each

independently tested as covariates. Sire (father) of ewe lamb was used as a random

effect.

Puberty and fertility data were analyzed using the generalized linear mixed

model procedures with a binomial distribution and logit link function (PROC GLIMMIX).

Fixed effects were dam source, dam age (years), birth type, age and live weight at the

start of the period (teasing for puberty; mating for fertility). Average daily gain (split into

teasing for puberty and mating for fertility), FAT, EMD, PWT, PEMD, PFAT, leptin or

ghrelin concentration (for puberty) or leptin or ß-hydroxybutyrate concentration (for

fertility) were each independently tested as covariates. Sire (father) of ewe lamb was

used as a random effect. Reproductive rate data was analyzed using the generalized

linear mixed model procedures with a multinomial distribution and logit link function

(PROC GLIMMIX). The same fixed effects, covariates and random effects were used

as the as for the analysis of fertility.

Blood metabolite data (leptin, ghrelin, ß-hydroxybutyrate) were analysed using

mixed models (PROC MIXED) allowing for repeated-measures and included, as fixed

effects, dam source, dam age (years), birth type and age and live weight at start of

teasing. FAT, EMD, PWT, PEMD or PFAT were each independently tested as

covariates. Identification number of the ewe lamb within sire (father) group was used as

a random effect. Mean metabolite concentrations were analyzed using analysis of

variance (repeated measures), where Factor A was hormone concentration and Factor

B was date at sampling (PROC ANOVA).

All 2-way interactions among the fixed effects were included in each model and non-

significant (P > 0.05) interactions were removed from the final model. The data for

44

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puberty, fertility and reproductive rate are presented as logit values and back-

transformed percentages.

3. Results 3.1. Live weight

Mean (± SEM) live weight increased from 38.2 ± 0.3 kg on Day –70 to 52.6 ±

0.4 kg on Day +98 (Fig. 2) and differed with age at the start of the teasing period (P <

0.001). On average, single-born lambs were heavier than twin-born lambs (37.6 vs

35.6 kg; P < 0.01) and single-reared lambs were heavier than twin-reared lambs (37.5

vs 35.7 kg; P < 0.001).

Fig. 2 Average live weight (± SEM) of Merino ewe lambs born as singles (black; n = 135) or

twins (white; n = 55). Day 0 is the day when entire rams were first introduced for the first mating

period. BS: blood sample.

The correlations among live weight, PWT, EMD, PEMD, FAT and PFAT are

shown in Table 1. Medium to strong positive correlations were observed between live

weight and FAT and EMD, PWT and EMD and PEMD and PFAT (Table 1).

During the teasing period, the ADG was 117 ± 2 g and differed with age at the

start of the teasing (P < 0.001), but was not affected by dam age (years) nor dam

source (P > 0.05). Ewe lambs born and reared as twins grew around 10 g/d faster than

ewe lambs born and reared as singles (P = 0.06; Table 2). During the mating period,

Teasing period

-70 -40 0 24 98

35

40

45

50

55

Live weight (Kg)

Day of experiment

Mating period 1

Mating period 2

BS BS BS

58 80

45

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the ADG was 58 ± 2 g and was affected by birth type (P < 0.05; Table 1), but the other

variables had no effect. Ewe lambs born and reared as a twin grew around 9 g/d faster

than ewe lambs born and reared as a single during this period (Table 2).

Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,

PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs.

Variable LW PWT EMD PEMD FAT PFAT

LW 1 0.79 0.70 0.24 0.52 0.26

PWT 0.79 1 0.63 0.29 0.33 0.11

EMD 0.70 0.63 1 0.76 0.54 0.52

PEMD 0.24 0.29 0.76 1 0.40 0.67

FAT 0.52 0.33 0.54 0.40 1 0.71

PFAT 0.26 0.11 0.52 0.67 0.71 1

Live weight during the experiment was positively related with EMD, FAT, PWT,

PEMD or PFAT (P < 0.001). Ewe lambs were 1.2 kg heavier as EMD increased by 1

mm, 2.1 kg heavier as FAT increased by 1 mm, 2.0 kg heavier as PWT increased by 1

kg, 1.9 kg heavier as PEMD increased by 1 mm, or 3.1 kg heavier as PFAT increased

by 1 mm. During the teasing period, ADG was positively correlated with EMD (P <

0.001), FAT (P < 0.01), PWT (P < 0.001) and PFAT (P ≤ 0.05), but not PEMD (P >

0.05). The ADG during this period increased by 4.8 g/d as EMD increased by 1 mm, by

7.2 g/d as FAT increased by 1 mm, by 10 g/d as PWT increased by 1 kg or 12.8 g/d as

PFAT increased by 1 mm. During the mating period, ADG was positively correlated

with PWT (P < 0.001) and increased by 5 g/d as PWT increased by 1 kg. It was not

related to EMD, FAT, PEMD or PFAT (P > 0.05).

3.2. Live weight and age at puberty

Of the 190 ewe lambs, 170 (89%) displayed oestrus during the ‘Teasing period’

and ‘Mating period 1’, and the proportion that displayed oestrus was not affected by

dam source, dam age (years), birth type or rear type (P > 0.05). Ewe lambs that were

heavier at the start of the teasing period (P < 0.001) and ewe lambs that grew faster

(high ADG) were more likely to achieve puberty (P < 0.05). Thus, on average, the

proportion of ewe lambs that displayed oestrus increased by 4% with each 20 g/d

increase in ADG during the ‘Teasing period’. As shown in table 2, ewe lambs were

more likely to attain puberty if they had high values for EMD (P < 0.001), FAT (P <

0.01), PWT (P < 0.001; Fig. 3), PEMD (P < 0.05) or PFAT (P < 0.05). After adjustment

for effects of live weight, the relationships with muscle and fat accumulation were no

longer evident. 46

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Table 2: Relationships among birth and rear type and the average daily gain (ADG; split into

teasing and mating periods), reproductive performance, and blood leptin concentrations in

Merino ewe lambs. LW = live weight.

Birth type Rear type

1 2 1 2

n Mean ±

SEM

n Mean ±

SEM

n Mean ±

SEM

n Mean ±

SEM

ADG during teasing (g/d)

135 114 ± 2a 55 123 ± 3a 140 114 ± 2a 50 124 ± 4a

ADG during mating (g/d)

135 56 ± 2a 55 65 ± 4b 140 56 ± 2a 50 64 ± 4a

Age at first oestrus (days)

121 198 ± 3a 49 196 ± 5a 125 199 ± 3a 45 193 ± 5a

LW at first oestrus (kg)

121 42 ± 0.5a 49 40 ± 0.7c 125 42 ± 0.5a 45 40 ± 0.8c

Puberty (%) 121 90a 49 89a 125 89a 45 90a

Age at pregnancy (days)

49 270 ± 4a 19 279 ± 7a 50 271 ± 4a 18 278 ± 7a

LW at pregnancy (kg)

49 46.7 ± 0.7a 19 47.3 ± 1.1a 50 46.7 ± 0.7a 18 47.4 ± 1.1a

Fertility (%) 49 36a 19 35a 50 36a 18 36a

Fecundity (%) 49 12a 19 37b 50 14a 18 34a

Reproductive rate a a a a

Leptin (ng/mL) 1.9 ± 0.02a 1.9 ± 0.03a 1.9 ± 0.02a 1.9 ± 0.04a ab values within columns with different superscripts are different (P < 0.05); ac values within columns with

different superscripts are different (P < 0.01)

At the first detected oestrus, the average age was 197 ± 2 days (range 147–

290) and the average live weight was 41.4 ± 0.4 kg (range 29.1–57.6), 67% of mature

body weight (estimated to be 61.6 ± 0.4 kg; range 46–79). Live weight at first oestrus

differed with birth type (P < 0.01), rear type (P < 0.05) and age at the start of the

‘Teasing period (P < 0.01), but neither birth type nor rear type were related to age at

first oestrus (P > 0.05; Table 2). On average, ewe lambs born and reared as a single

were heavier by 2 kg (P < 0.05) and older by 6 days at first oestrus than those reared

as a twin (P > 0.05; Table 2).

Live weight at first oestrus was positively related with increases in EMD (P <

0.001), FAT (P < 0.001), PWT (P < 0.001), PEMD (P < 0.01) and PFAT (P < 0.01)

(Table 3). Thus, live weight at first oestrus increased by 1.3 kg as EMD increased by 1

mm, 2.2 kg as FAT increased by 1 mm, 2.3 kg as PWT increased by 1 kg, 2.2 kg as

PEMD increased by 1 mm and 2.6 kg as PFAT increased by 1 mm. Age at first oestrus

was not related to any of these traits (P > 0.05; Table 3).

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Table 3: Outcomes of statistical analyses of the relationships among live weight (LW) or age at

start of the “teasing” or “mating” periods, average daily gain (ADG) split into “teasing” and total

“mating” periods, phenotypic values for eye muscle depth (EMD) and fatness (FAT), ASBV for

post-weaning weight (PWT), post-weaning eye muscle depth (PEMD), post-weaning fatness

(PFAT), and blood metabolite concentrations, and reproductive performance and leptin

concentration in Merino ewe lambs. Information from both mating periods was combined for

fertility, fecundity and reproductive rate. Age (1st

oestrus) LW (1st

oestrus) Puberty Fertility Fecundity Reproductive

Rate Leptin

Variable Days Kg % % % % ng/mL

Age (teasers in) NA ** NS NA NA NA * LW (Teasers in) NS NA ** NA NA NA *** LW (Rams in) NA NA NA *** * *** NA

ADG (Teasing) NS NA * NA NA NA NA

ADG (Total Mating)

NA NA NA * NS NS NA

PWT NS *** *** *** * *** *** PEMD NS ** * NS NS NS *** PEMD (+PWT) NS NA NS NS NS NS * PFAT NS ** * NS NS NS *** PFAT (+PWT) NS NA NS NS NS NS *** EMD NS *** *** ** * *** *** EMD (+LW at scan)

NS NA NS NS NS NS ***

FAT NS *** ** * NS * *** FAT (+LW at scan)

NS NA NS NS NS NS ***

Plasma Ghrelin NS * NS NA NA NA NA

Plasma ß-Hydroxybutyrate

NA NA NA NS NS NS NA

Plasma Leptin NS * * NS NS NS NA

Plasma Leptin (+LW at scan)

NS NA NS NS NS NS NA

P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05; NA, not applicable

48

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Fig. 3. The relationship between the Australian Sheep Breeding Value (ASBV) for post-weaning

weight (PWT) and the proportions of Merino ewe lambs that (A) achieved puberty by 251 days

of age (P < 0.001) and (B) were pregnant after mating at age 7-11 months (P < 0.001). Data

from single and twin births were retained in the model and the relationship was pooled within

the birth-type classes. The broken lines represent upper and lower 95% confidence limits.

3.3. Fertility, fecundity and reproductive rate

A total of 68 of 190 (36%) ewe lambs were pregnant with 80% carrying a single

lamb and 20% carrying twin lambs. Ewe lambs that were heavier at the start of the

mating period were more fertile (P < 0.001; Fig. 4) and fecund (P < 0.01) than lighter

animals. The most significant increases in fertility rate occurred when the ewe lambs

weighed more than 40 kg at start of mating (Fig. 4).

0

20

40

60

80

100Puberty reached (%)

0

20

40

60

80

1 2 3 4 5 6 7 8 9ASBV for Post-weaning weight (PWT; Kg)

Fertility (%)

A

B

49

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Fig. 4. Relationships between live weight at the start of mating and (A) fertility (P < 0.001) and

(B) reproductive rate (number of lambs in utero per 100 ewes mated; P < 0.001) in Merino ewe

lambs mated at 8-11 months of age. Data from both mating periods and are combined. Data

from single and twin birth were retained in the model and the relationship was pooled within the

birth-type classes. The broken lines represent upper and lower 95% confidence limits.

At the estimated date of conception, live weight was 46.9 ± 0.6 kg (range 37.6–58.1)

and age was 273 ± 4 days (range 215–321). The ADG during the “Mating period” was

positively related to fertility (P < 0.05) but not fecundity (P > 0.05). By contrast, birth

type influenced fecundity (P < 0.05) but not fertility (P > 0.05; Table 2). Neither fertility

nor fecundity were influenced by dam age (years), dam source or rear type (P > 0.05).

On average, ewe lambs born and reared as twins were 9 days older and 0.7 kg heavier

at the estimated date of conception than ewe lambs born as singles (P > 0.05; Table

2).

Fertility (%)

Reproductive rate (%)

Live weight at start of mating (Kg)

0

20

40

60

80

100

120

140

35 40 45 50

0

20

40

60

80A

B

50

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Reproductive rate was positively related to live weight at the start of mating (P <

0.001; Fig. 4). The relationship was linear with each extra kg of live weight at the start

of mating associated with an extra 4.5 fetuses per 100 ewes mated (Fig. 4). Birth type,

rear type, dam age and dam source had no effect on reproductive rate (P > 0.05).

Fertility was positively correlated with EMD (P < 0.01), FAT (P < 0.05) and PWT

(P < 0.001; Fig. 3), but not PEMD or PFAT (Table 2). Fecundity was positively related

with EMD (P < 0.05) and PWT (P ≤ 0.05), but was not with FAT, PEMD or PFAT (P >

0.05; Table 3). Reproductive rate was positively correlated with EMD (P < 0.001), FAT

(P < 0.05) and PWT (P < 0.001), but not with PEMD or PFAT (P > 0.05; Table 3). After

adjustment for effects of live weight, the effects of muscle and fat accumulation on

fertility, fecundity and reproductive rate were no longer evident. 3.4. Hormone and metabolite profiles

The concentration of leptin increased gradually between November 2010 and

March 2011 from 2.01 ± 0.02 to 2.12 ± 0.03 ng/mL, the exception being a lower

concentration in the blood sample collected during February 2011 (Day 0; 1.2 ± 0.02

ng/mL) In contrast, ß-hydroxybutyrate concentrations decreased from 0.38 ± 0.009 to

0.33 ± 0.008 mmol/L, the exception being a higher value in the sample collected during

February 2011 (Day 0; 0.59 ± 0.12 mmol/L). Leptin concentration was positively related

to the proportion of ewe lambs that attained puberty (P ≤ 0.05) and live weight at first

oestrus (P < 0.01), whereas ghrelin concentration was negatively related to live weight

at first oestrus (P < 0.05). Leptin and ghrelin concentrations were not related to age at

first oestrus and ghrelin concentration was not related to the proportion of ewe lambs

that attained puberty (P > 0.05). Neither leptin nor ß-hydroxybutyrate concentrations

were associated with fertility, fecundity or reproductive rate (P > 0.05; Table 3). Leptin

values differed with birth type (P < 0.05; Table 1), but not with rear type, dam source or

dam age (P > 0.05). Leptin concentration was positively related to increases in PWT (P

< 0.001), PEMD (P < 0.001), PFAT (P < 0.001), EMD (P < 0.001) and FAT (P < 0.001)

(Table 2). Leptin concentration increased by 0.05 ng/mL as PWT increased 1 kg, 0.24

ng/mL as PEMD increased 1 mm, 0.36 ng/mL as PFAT increased 1 mm, 0.05 ng/mL

as EMD increased 1 mm or 0.11 ng/mL as FAT increased 1 mm. The relationships

between leptin and the accumulation of muscle and fat were still evident when live

weight was included in the statistical analysis. Neither ghrelin nor ß-hydroxybutyrate

concentrations were related to phenotypic or genotypic values for growth, fat or muscle

(P > 0.05).

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4. Discussion

Most Merino ewe lambs achieved puberty by 250 days of age when their

average live weight was around 41 kg, or about 67% of their estimated mature live

weight, within the range of 50-70% that has previously been considered as the

threshold for spontaneous puberty in sheep [reviewed by 1,2]. Ewe lambs with higher

breeding values for growth were more likely to achieve puberty at around 250 days of

age. They were heavier at the start of teasing and grew faster during the teasing

period, and both of these factors were statistically related to the percentage of ewes

achieving puberty by this age. This observation is consistent with previous reports that

faster growth results in more ewes achieving puberty at a younger age in female sheep

[7, 19]. In the current study, more than 90% of ewe lambs with a PWT greater than 5

achieved puberty by this age, whereas the value was less than 40% for lambs with a

PWT of 1, close to the average for Merino ewes on the National Sheep Genetics

database (www.sheepgenetics.org.au). Early puberty also led to first mating at a

younger age and would allow second mating at 20 months of age and ready integration

with the adult ewe flock. Thus, overall, there are many advantages to selection of

Merino ewe lambs with a high breeding value for growth.

Fertility also improved as breeding value for growth increased, reflecting

previous reports of a positive genetic correlation between weaning weight, rapid growth

and reproductive performance in young female sheep [20, 21]. Ewe lambs with higher

breeding values for growth were heavier at the start of mating, grew faster during

mating and had higher fertility, as previously observed [7, 22]. The relationship

between live weight at the start of mating and fertility rate was linear over the range

40–55 kg. High breeding values for growth and live weight at the start of mating were

also associated with improved fecundity and overall reproductive rate. Higher live

weight at the start of mating may provide a greater benefit for ewe lambs than for

mature ewes, because for each extra kg, there was a gain of 4.5-4.8 extra fetuses per

100 ewe lambs mated, in contrast to only 1.7-2.4 extra fetuses for mature ewes [23].

While the ewe lambs own breeding values for growth include both phenotypic and

genetic components, we have recently studied larger data sets and found that the

breeding value for growth of the sire of Merino ewe lambs is significantly related to the

reproductive performance of their daughters when they mated at 8-10 months of age

(A.N. Thompson, 2013, unpublished data). We are therefore able to conclude that

genetic strategies, as well as nutritional management, can be used improve

reproductive rate in Merino ewe lambs.

In the present study, the lambs that were still pregnant at scanning were

estimated to have conceived about 80 days after achieving puberty, during which time

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they had gained more than 5 kg in live weight. Depending on cycle length, which vary

from 13–19 d and average 17 d [reviewed by 24], it seems likely that on average they

conceived on their fourth or fifth cycle post-puberty. This deduction, together with the

observation that more 90% of ewe lambs appeared to achieve puberty and yet only

36% were scanned as pregnant, suggests poor conception or a high incidence of

embryo mortality in the present experiment. Hare and Bryant [25] found that the

proportion of ewe lambs with living embryos was greater in those that were mated at

their third oestrus, intermediate in those mated at their second oestrus, and lowest in

those mated at their pubertal oestrus. Ewe lambs born as singles were about 2 kg

heavier, but surprisingly 2 days older, at their first oestrus than their twin-born

counterparts, whereas those born as singles were younger and lighter at the estimated

date of conception. Indeed, ewe lambs that were born as a twin gained 7.7 kg between

puberty and conception compared to 4.6 kg for those born as singles which, despite

some evidence of compensatory growth, indicates that the oestrous cycle from which

pregnancy was maintained was later than for those born as singles. These

observations further highlight the importance of achieving puberty as early as possible

and also suggest that twin-born lambs may benefit from either a delayed mating after

teasing or a longer mating period.

This and other experiments indicate significant variation in the relationship

between live weight at mating and fertility and reproductive rate. Fertility, especially

under field conditions, is always lower and more variable for ewe lambs than for mature

ewes [reviewed by 26], suggesting important effects of unidentified and thus

uncontrolled environmental factors. In the present study, the animals experienced high

environmental temperatures (above 32º C) before and during mating, a known cause of

infertility in Merino ewes [27]. A second possibility is variation in metabolic status, as

described below – we did not limit nutrition but the decline in the trajectory of weight

gain during the week before the start of Mating period 1, followed by a low ADG during

the mating period, suggest that negative energy balance may have prevented the

continuation of ovulation or compromised fertility [reviewed by 28, 29]. This did not

happen in our previous study where the ADG during mating was over 200 g/d and 75%

of the ewe lambs conceived [7]. Further research is needed to test this hypothesis.

The depths of muscle and fat were related to live weight at puberty, and to

fertility, fecundity and reproductive rate. These relationships were revealed when live

weight was excluded in the model. This can be explained by the correlations among

these traits that were observed in this experiment. Obviously, live weight includes

muscle and fat and thus raises questions of interpretation. An important consideration

is that live weight per se is simply mass and so encompasses no physiological or

mechanistic process that would affect the reproductive system. On the other hand,

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tissues that are metabolically, physiologically and hormonally active, such as muscle

and fat, can become involved in control processes at the brain, pituitary, ovarian or

uterine levels. Moreover, the activities of muscle and fat will be affected by nutrition,

whether or not liveweight responses are detectable. Certainly, these processes could

explain differences in outcomes between ad libitum feeding [7] and limited diets

(present study), but confirmation of these using larger data sets is still required.

For adipose tissue, leptin is the likely endocrine signal to the reproductive

system and it has shown to affect directly the neuroendocrine processes that control

the initiation of puberty in female sheep [reviewed by 30]. The present observations

support this concept because leptin concentration was positively correlated with

increases with FAT, PWT and PFAT. For muscle, however, endocrine links to the

reproductive control centres have not been clearly identified. Interestingly, we found

that leptin concentration was positively correlated with increases with EMD and PEMD,

extending on the observations of Wang et al. [31], and raising the possibility that leptin

from intramuscular fat can play a role in the control of the reproductive axis. This needs

to be tested in further research.

Leptin and ghrelin concentrations were not related to age at first oestrus, but

puberty and live weight at first oestrus was positively correlated with leptin

concentration and negatively with ghrelin concentration. These findings align with the

principle that, under favourable metabolic conditions, we can expect an increase in

leptin concentration and a decline in ghrelin concentration because ghrelin plays a

dominant inhibitory role whereas leptin has a promotable effect on the reproductive

axis [12, reviewed by 14]. The variability in live weight observed during the mating

period was reflected in a decrease in leptin concentration in the sample collected one

week before the mating period. We would expect the physiological factor to be more

responsive than live weight to acute changes in energy balance, so we can surmise

that the ewe lambs were in negative balance at the start of the mating period, and this

is supported by the observation of high ß-hydroxybutyrate levels. These circumstances

could explain the relatively poor reproductive performance in the current experiment

compared to our previous study [7]. Leptin and ß-hydroxybutyrate concentrations were

not associated with fertility, fecundity or reproductive rate. On the other hand, leptin

concentrations were positively associated with higher values for growth, muscle and fat

accumulation, and therefore an improvement in reproductive performance.

4.1. Conclusion

In conclusion, Merino ewe lambs with higher breeding values for growth will

achieve puberty earlier and express higher levels of fertility and fecundity. Live weight

at start of mating plays an important role in the reproductive performance of ewe

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lambs, and muscle and fat both appear to be physiologically active components of the

body mass because phenotypic or breeding values that increase their rate of

accumulation are associated with improved reproductive performance. At this stage,

the physiological factors in these tissues that are responsible for the reproductive

outcomes are not clear, but it seems feasible that intramuscular fat, and the leptin that

it might produce, could be a determinant of reproductive performance. Further research

is required to determine the ADG values, before and during the mating period, that

would guarantee reproductive success in ewe lambs under extensive conditions.

Acknowledgements

The authors wish to thank to Margaret Blackberry (University of Western Australia) for

her assistance with the hormone analyses, and Kristy Robertson for her assistance in

the care and management of the animals. During his doctoral studies, Cesar Rosales

Nieto was supported by Mexico’s National Council for Science and Technology

(CONACyT), the Department of Agriculture and Food of Western Australia, the

Cooperative Research Centre for Sheep Industry Innovation, and the UWA School of

Animal Biology.

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[14] Tena-Sempere M. Ghrelin as a pleotrophic modulator of gonadal function and

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[18] SAS Institute. 2008. SAS/Stat User’s guide, Version 9.2. SAS Institute Inc., Cary,

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[20] Barlow R, Hodges C. Reproductive performance of ewe lambs: genetic correlation

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[22] McGuirk BJ, Bell AK, Smith MD. The effect of bodyweight at joining on the

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[26] Quirke JF. Regulation of puberty and reproduction in female lambs: A review.

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[27] Sawyer G. The influence of radiant heat load on reproduction in the Merino ewe. I.

The effect of timing and duration of heating. Australian Journal of Agricultural

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[28] Scaramuzzi RJ, Campbell BK, Downing JA, Kendall NR, Khalid M, Muñoz-

Gutiérrez M, et al. A review of the effects of supplementary nutrition in the ewe on

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that regulate folliculogenesis and ovulation rate. Reproduction Nutrition

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[29] Marai IFM, El-Darawany AA, Fadiel A, Abdel-Hafez MAM. Physiological traits as

affected by heat stress in sheep—A review. Small Ruminant Research. 2007;71:1-

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[30] Foster DL, Nagatani S. Physiological Perspectives on Leptin as a Regulator of

Reproduction: Role in Timing Puberty. Biology of Reproduction. 1999;60:205-15.

[31] Wang J, Liu R, Hawkins M, Barzilai N, Rossetti L. A nutrient-sensing pathway

regulates leptin gene expression in muscle and fat. Nature. 1998;393:684-8.

57

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Chapter 3

Prepubertal growth and muscle and fat accumulation in male and female sheep – relationships with metabolic hormone concentrations, timing of

puberty, and reproductive performance

C.A. Rosales Nietoa,b,c, M.B. Fergusona,b,d,f, J.R. Briegelb, M.P. Hedgere, G.B. Martinc,g, A.N.

Thompsona,b,d

aCRC for Sheep Industry Innovation and the University of New England, Armidale, NSW, 2351,

Australia bDepartment of Agriculture and Food of Western Australia, South Perth, WA 6151, Australia

cThe UWA Institute of Agriculture, University of Western Australia, Crawley, WA 6009, Australia dSchool of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150,

Australia eMonash Institute of Medical Research, Monash University, Vic 3168, Australia

fPresent address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch 8024,

New Zealand gNuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford OX3 9DU,

UK

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Abstract Across a variety of species, metabolic homeostasis is aligned with changes in growth

and body composition, through processes mediated by circulating metabolites and

metabolic hormones, and it is eventually linked to reproductive success. In the present

study, with young Merino sheep, we determined the effects of differences in the rates

of growth and muscle and fat accumulation on the circulating concentrations of

metabolites and metabolic hormones, and the relationships between these factors and

the timing of puberty and ensuing reproductive performance. We used 64 females and

62 males with known phenotypic values for depth of eye muscle (EMD) and fat (FAT)

and known Australian Sheep Breeding Values at post–weaning age (220 days) for live

weight (PWT), depth of eye muscle (PEMD) and depth of fat (PFAT). Blood was

sampled every 20 min for 8 h via jugular cannula and assayed for growth hormone

(GH), insulin-like growth factor I (IGF-I), insulin, leptin, ghrelin, follistatin, glucose and

non-esterified fatty acids (NEFA). In males, the only relationships detected were the

positive effects of PWT on the concentrations of GH, follistatin, and glucose, the

positive effects of FAT and PFAT on IGF-I concentrations (P < 0.01). An unexpected

finding was the negative relationships between testosterone concentrations and

muscle variables (P < 0.001) and PFAT (P < 0.05). In females, the only relationship

detected was the positive effect of EMD on insulin concentrations (P < 0.05).

Reproductive variables were measured only in females. Live weight at first oestrus was

related positively to insulin concentration and negatively to GH concentrations (P <

0.05). No other relationships with reproductive variables were significant. The single

day of sampling appears to have limited the opportunities for detecting the

relationships hypothesized, but those relationships that were detected suggest subtle

differences between the sexes in the effects of changing the rates of growth and

muscle and fat accumulation on metabolic homeostasis, perhaps due to interference

by testosterone.

Introduction

The rates of growth and accumulation of muscle and adipose tissues are controlled by

a variety of regulatory and differentiation processes that are affected by many factors,

including nutrition, age, gender, mature body size, genetics, and health. Most of these

interactions are regulated by the endocrine system (Berg and Butterfield 1968; Owens

et al., 1993; Hocquett et al., 2010). For muscle growth and development, the major

hormones are growth hormone (GH), insulin-like growth factor (IGF-I), insulin, leptin,

and follistatin. In males, the anabolic effects of androgens add an extra dimension to

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the control of muscle accumulation (Schanbacher et al., 1980; reviewed by Etherton

1982; Spencer 1985; Lee and McPherron 2001; Zeidan et al. 2005; Velloso et al.,

2008). For adipose tissue, the critical factors are leptin and non-esterified fatty acids

(NEFA), both of which are implicated in fat accumulation and mobilization. Importantly,

muscle and adipose tissue are not independent but interact with each other through

glucose/insulin, ghrelin and leptin (Kokta et al., 2004; Patel et al., 2006; Barazzoni et

al., 2007; DeFronzo and Tripathy 2009).

Interactions among these factors inform the central nervous system about the

status of body reserves, to which the metabolic control centres change appetite,

metabolic rate, body temperature and behaviour. In parallel, the brain responds to

information on the status of body reserves to implement strategies for reproduction

(Blache et al., 2007). The relationship between the reproductive and metabolic systems

is mediated, at least in part, by physiological signals from metabolic tissues to the

reproductive control centres and tissues, affecting sexual maturation and subsequent

reproductive performance (review: Martin et al. 2009). In a favourable metabolic

environment, the onset of puberty can be promoted by GH, IGF-I, leptin and

testosterone (Bourguignon 1988; Roberts et al., 1990; Foster and Nagatani, 1999;

Wheaton and Godfrey, 2003) whereas, in an unfavourable metabolic environment,

puberty can be delayed by deficiencies in GH, insulin or glucose and high

concentrations of ghrelin and NEFA (Advis et al., 1981; Richards et al., 1989; Bucholtz

et al., 2000; Tena-Sempere, 2007). Additionally, after puberty, fertility can be

modulated by leptin and perhaps follistatin (Smith et al., 2002; Jorgez et al., 2004;

Kimura et al., 2010).

Therefore, we would expect these blood-borne signals to respond to factors,

both environmental and genetic, that affect reproductive success by changing the rates

of body growth and accumulation of muscle and adipose tissue. We tested this

hypothesis in lambs by measuring the concentrations of GH, IGF-I, insulin, glucose,

leptin, ghrelin, follistatin and NEFA and assessing the relationships with variables that

reflect potential for growth and muscle and fat accumulation. In females, we also

assessed the relationships with the onset of puberty and reproductive performance.

Material and Methods This work was done in accordance with the Australian Code of Practice for the Care

and Use of Animals for Scientific Purposes (7th Edition, 2004) and was approved by

the Animal Ethics Committees of both the University of Western Australia and the

Department of Agriculture and Food (Western Australia).

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Experimental location and animal management

Merino lambs (n = 380) were born in June 2010 (Day 0) on the research farm of the

University of Western Australia (32.2° S, 115.8° E). The dams of the lambs had been

sourced from two ram breeding flocks in Western Australian (‘Merinotech WA’ and

‘Moojepin’) and the sires had a wide range in Australian Sheep Breeding Values

(ASBV) for growth, muscle and fat. The Australian Sheep Breeding Values delivered by

MERINOSELECT are the result of collation and analysis of individual performance

values, pedigree information and relevant environmental and management information

of the animals from participating breeders. Data for birth date, birth weight and birth

type were collected for the lambs.

Ewe lambs

In November 2010 (Day 139), 190 ewe lambs were transported to the Medina

Research Station (32.2°S, 115.8°E) where they were allocated among eight groups,

each of equal average live weight upon arrival, and each held in a separate indoor pen

(6 x 7 m). Upon arrival, animals were treated with a broad spectrum oral anthelmintic

(12.5 mL/head; Triton®, Merial Australia, Parramatta, NSW), vaccinated against

clostridial disease, and injected with selenium (1 mL/head; Glanvac™ 6S Vaccine,

Pfizer Australia, West Ryde NSW), vitamin B1 (2 mL/head; Thiamine Hydrochloride

[125mg/mL], Nature Vet, Glenorie, NSW) and vitamins A, D and E (1 mL/head; Vit A

500,000 iu; Vit D3 75,000 iu, Vit E 50 iu/mL; Vet ADE®, Auckland, New Zealand). The

animals had ad libitum access to water and to dietary pellets that were introduced over

a 7-day period. The pellets were based on barley, wheat and lupin grains, cereal straw

and hay, canola meal, minerals and vitamins, and were formulated to provide 11.5 MJ

metabolisable energy per kilogram of matter, 15% protein and minerals, thus meeting

the theoretical daily requirements for maximum growth (Macco Feeds Australia).

On November 30 (Day 168), when ewe lambs were on average 157 days old

(range 136-176) and weighed 36.2 ± 0.3 kg (range 24.8-50.8), a vasectomized Merino

ram with a marking harness (MatingMark®; Hamilton, NZ) was introduced into each

pen to detect the onset of oestrus. The crayons on the harnesses were changed every

two weeks. On 29 of December of 2010 (Day 197), 64 of the ewe lambs were selected

on the basis of sire, birth type and live weight, and were transported to the animal

house at Shenton Park Field Station (31.9 °S, 115.8 °E). Ewe lambs were on average

186 days old (range 165 to 205) and weighed 42.4 ± 0.4 kg (range 34 to 49.5).

Females were placed in individual pens (1.0 x 1.78 x 0.78 m) to which they were

acclimatized for 14 days before blood sampling. Each animal had access to clean

water and an amount of the pellets (described above) that resulted in residues between

100 and 200 g per day (approximately 10%).

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On January 14 (Day 213), ewe lambs were returned to the university farm and

combined with the remainder of the ewe lamb flock and vasectomized rams (Figure 1)

in a 30 x 120 m plot where they had access to clean water, ad libitum oaten hay and

300 g of lupins/head daily. Crayon marks on the rumps were recorded three times per

week at Medina and once per week on the farm to estimate the date of first standing

oestrus. Crayon marks were scored (1, 2 or 3), with Score 1 being one narrow mark on

the middle or the edge of the rump and Score 3 as being one big wide mark on the

rump. Only marks with Score 2 or 3 were accepted as indicating oestrus. When the first

Score 2-3 crayon mark was recorded, the date and ewe lamb age were noted as an

indication of the onset of puberty. The date was deemed to be age at first oestrus and

the closest LW recorded to that date was deemed to be live weight at first oestrus.

The vasectomized rams were removed on February 8 (Day 238) and ewe

lambs were allocated on the basis of live weight and sire into 8 groups of 24. An

experienced ram with a marking harness (MatingMark®; Hamilton, NZ) was introduced

into each group to begin the “Mating period” (Figure 1) when the ewe lambs were on

average 226 days old (range 206-246) and weighed 42.4 ± 0.3 kg (range 24.3-56.4).

Figure 1. Experimental protocol for ewe and ram lambs.

Animals were fed ad libitum oaten hay and 300 g/head daily of lupin grain. The rams

were removed after 46 days. Pregnancy and the number of fetuses were determined

by ultrasound scanning in mid-April and mid-June and the data was used to generate

values for fertility and reproductive rate. 62

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The ewe lambs were weighed every week and these liveweight data were used

to detect the live weight at the onset of puberty and pregnancy. The depths of the

longissimus dorsi muscle and subcutaneous fat at a point 45 mm from the midline over

the twelfth rib were measured using ultrasound by an accredited specialist when the

ewe lambs were on average 167 (range 146-186) and 218 (range 198-228) days old.

Over both measurements, the range in eye muscle depth (EMD) was 20-33 mm and

the range in C-site fat depth (FAT) was 2-8 mm. The ultrasound data combined with

the genetic information of the ewe (dam, sire, birth weight, birth type) were used to

generate ASBVs at post-weaning age for weight (PWT; range 0-9 kg), depth of eye

muscle (PEMD; range 0.0-2.6 mm) and fat (PFAT; range 0.0-1.2 mm) using

MERINOSELECT (Brown et al., 2007).

Ram lambs

On 22 December 2010 (Day 190), ram lambs (n = 190) were transported to Medina

Research station (32.2° S, 115.8° E) where they were allocated among 15 groups of

equal average live weight (on the basis of live weight upon arrival), each held in a

separate indoor pen (6 x 7 m). Upon arrival, animals were also subjected to the health

routine described above for the ewe lambs. The animals had ad libitum access to water

and to sheep pellets (described above) that were introduced over a 7-day period.

On 16 March 2011 (Day 274), 64 ram lambs were selected on the basis of sire,

birth type and live weight and transported to the animal house in Medina research

station. They were 264 days old (range 245 to 282) and 56 ± 0.5 kg (range 46 to 62)

and were placed in individual pens (1.0 x 1.5 x 0.6 m), where they were acclimatized

for 14 days. They had access to clean water and pellets as described for the ewe

lambs. Two ram lambs with very nervous temperament were not subjected to blood

sampling but remained in their pens. After the experiment, the animals returned to the

ram lamb flock at the Medina research station.

Live weight was recorded weekly. The depths of the longissimus dorsi muscle

and subcutaneous fat, at a point 45 mm from the midline over the twelfth rib, were

measured using ultrasound by an accredited specialist when the animals were 211

(range 201-217) days old. Over both measurements, the range in eye muscle depth

(EMD) was 20-31 mm and the range in C-site fat depth (FAT) was 2-6 mm. As for the

ewe lambs, the ultrasound data and genetic information were used to generate ASBVs

at post-weaning age for PWT, PEMD and PFAT (Brown et al., 2007).

Blood sampling and immunoassay

The lambs were fitted with jugular catheters at least 18 h before sample collection.

Feeders were removed 3 hours before sampling began. Blood was sampled every 20

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min for 8 h and 5mL of every sample was placed into heparinised tubes. In addition, 3

mL of samples 4, 7, 10, 13, 16, 19 and 22 were also placed into flouride/oxalate tubes,

and 2 mL of samples 7, 10, 13 and 16 were placed into EDTA tubes. Samples were

placed immediately on ice and then centrifuged at 2000 g for 20 min so plasma could

be harvested. Samples with heparine and fluoride/oxalate were stored at –20 ºC and

samples in EDTA were immersed in liquid nitrogen then stored at –80°C.

All 25 samples with heparin were assayed for growth hormone (GH) and

subsamples from all samples were pooled for each animal for assay of insulin-like

growth factor–I (IGF-I), insulin, follistatin, leptin, ghrelin. Testosterone was also

assayed for the ram lambs. Samples with EDTA were assayed for non-esterified fatty

acids (NEFA) and those with flouride/oxalate were assayed for glucose.

Growth hormone was assayed in duplicate 100 μL samples by double-antibody

radioimmunoassay (RIA), as described by Boukhliq et al. (1997). The limit of detection

was 0.05 ng/mL and the intra-assay CV was 19% at 1.05 ng/mL, 7.1% at 3.22 ng/mL,

and 2.3% at 7.92 ng/mL.

Insulin was assayed in duplicate 100 μL sample by double-antibody RIA, as

describe by Miller et al. (1995). The limit of detection was 0.04 μU/mL and the intra-

assay CV was 7% at 1.97 μU /mL, 7.3% at 1.98 μU /mL, and 2.3% at 12.9 μU /mL.

Plasma ghrelin was assayed in duplicate 100 μL samples by a modified double-

antibody RIA, as described by Miller et al. (2009). The limit of detection was 49 pg/mL

and the intra-assay CV was 6% at 94.3 pg/mL, 2.6% at 327 pg/mL, and 1% at 1549.9

pg/mL.

Leptin was assayed in duplicate 100 μL samples by double antibody RIA, as

described by Blache et al. (2000). The limit of detection was 0.05 ng/mL and the intra-

assay CV was 16% at 0.47 ng/mL, 3.3% at 1.10 ng/mL, and 3.6% at 1.79 ng/mL.

Plasma IGF-I was assayed in duplicate samples using the RIA described by

Gluckman et al. (1983). Interference by binding proteins was minimized by acid–

ethanol cryoprecipitation, as validated for ruminants by Breier et al. (1991). The limit of

detection for the assay was 0.05 ng/mL and the intra-assay coefficient of variation was

7% at 0.29 ng/mL and 5.1 % at 2.9 ng/mL.

Follistatin was measured in duplicate 100 μL samples with an RIA that

measures both free and bound forms, as previously described by Klein et al., (1991).

The assay employs purified heterologous bovine follistatin as standard and uses

iodinated bovine follistatin as tracer, as previously described (Robertson et al., 1987).

The limit of detection was 1.16 ng/mL and the intra- and inter-assay CV were 7.9% and

7.8%, respectively.

Testosterone was measured after extraction in a modified version of an in-

house RIA. All the samples and reagents were diluted in 0.01M Phosphate buffered

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saline containing 0.1% Gelatin (GPBS) unless otherwise stated. The standards, a stock

solution of testosterone (4-androsten-17ß-ol-3-one; Sigma Chemicals Co. Batch 108T-

0777) was prepared in ethanol. A substock of 24.1 ng/mL was made by diluting the

stock and stored at -20˚C. Standards were made by serial dilution to the following

concentrations: 0.049, 0.098, 0.187, 0.375, 0.75, 1.51, 3.02, 6.05, 12.1 and 24.2 ng/mL

in ethanol. The antiserum against testosterone-3 BSA (20C-CR2140R) was purchased

from Fitzgerald Industries International, diluted to 1:100 and stored at –20˚C. Cross-

reactions were 100% with testosterone, 0.5% with androstenedione and 0.01% with

DHEA. The tracer (1,2,6,7-)3H-testosterone (Amersham) with a specific activity of 90

Ci/mM was diluted in GPBS to give 15000 cpm per 100 µL. The assay included one

standard curve and up to 250 unknown samples in duplicate. The standard curve also

included triplicate tubes for total counts (TC) and NSB, 10 replicates of zero standard,

3 replicates of each standard, 6 replicates each of three quality control pools and 3

replicates of the solvent as a blank. On Day 1, 25 µL of unknown plasma and were

extracted with 2 mL AR diethyl ether. The extracts were poured into fresh 10 x 75 mm

glass tubes. The extracts and 25 µL of standards were dried and redissolved in 0.2 mL

with GPBS. The tracer (100 µL) and the antibody (100 µL, diluted 1:50,000) were

subsequently added to all tubes except total counts and NSB which received 0.1 mL

GPBS instead of the antibody. The tubes were mixed and incubated at 4˚C for 48 h.

On Day 3, normal rabbit serum (100 µL, 1:800) was added, followed by 100 µl second

antibody (anti-rabbit serum; 1:60 in GPBS containing 0.1M EDTA). The tubes were

mixed and incubated overnight at 4˚C. On Day 4, 2 mL of 2% polyethylene glycol (PEG

6000) in GPBS was added to all tubes (except TCs). The tubes were centrifuged in a

refrigerated (5˚C) centrifuge at 1500 g for 25 minutes, the supernatant aspirated and

the pellet was redissolved in 500 µL 0.05M HCl. The solution was dispensed into

counting vials and then mixed with 2 mL scintillant (Starcint, Packard Chemicals

Operations). The vials were capped, shaken and left in the dark for two hours before

counting in a liquid scintillation counter (Packard Tri Carb 1500). The limit of detection

was 0.02 ng/mL and the intra-assay CV was 11.6% at 0.28 ng/mL, 14.4% at 2.18

ng/mL, and 14.7% at 0.14 ng/mL.

An automatic analyser (AU400, Olympus, Tokyo, Japan) was used to measure

glucose using the glucose reagent kit supplied by Beckman Coulter (Gladesville NSW,

Australia), and to measure NEFA using the WAKO NEFA kit supplied by Novachem

(Collingwood, Victoria, Australia).

Data analysis

Data for the ewes and rams were analyzed separately using SAS version 9.3 (SAS

Institute Inc., Cary, NC, USA).

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Live weight and age at first oestrus, measures and ASBVs for body

composition, and mean concentrations for hormones and metabolites, were all

analysed using mixed models (Proc Mixed) and included fixed effects: dam source,

dam age (years), birth-reared type and age at blood sampling. Mean concentrations of

hormones and metabolites were each independently tested as a covariate and sire

(father) of the ewe lambs was used as a random effect.

Puberty and fertility were analyzed using the generalized linear mixed model

procedures with a binomial distribution and logit link function (PROC GLIMMIX). Fixed

effects were dam source, dam age (years) and birth-reared type. Insulin, leptin, IGF-I,

follistatin, ghrelin, mean GH, mean NEFA or mean glucose was each independently

tested as a covariate and sire (father) of the ewe lambs was used as a random effect.

Reproductive rate was analyzed using the generalized linear mixed model procedures

with a multinomial distribution and logit link function (PROC GLIMMIX). The same fixed

effects, covariates and random effects were used as the as the fertility analysis.

Mean hormone concentration was analyzed using the PROC GLM procedure.

Where factor A was hormone concentration and factor B was time of sampling, birth-

reared type, dam age (years), dam source or reproductive state (puberty attained or

not; pregnant or not; pregnant with single or twins) of the ewe lamb.

The relationships among PWT, PEMD, PFAT, EMD, FAT, GH, IGF-I, insulin,

leptin, ghrelin, follistatin, glucose, NEFA and testosterone were computed using

Pearson’s correlation, which is considered appropriate for parametric measures of

linear relationships between two variables, and Fisher’s transformation which helped to

derive confidence limits using PROC CORR.

All 2-way interactions among the fixed effects were included in each model and

non-significant (P > 0.05) interactions were removed from the final model. The data for

puberty, fertility and reproductive rate are presented as logit values and back-

transformed percentages.

Results

Growth

In females, PWT was not related to the concentrations of any of the blood factors

measured. In males, PWT was also not related to concentrations of IGF-I, insulin,

ghrelin, leptin or NEFA, but was strongly positively correlated to the concentrations of

GH and glucose and weakly positively correlated to follistatin (Table 1). For

reproductive function in females (Table 2), a strong positive relationship between PWT

and live weight at first oestrus was observed. Thus, higher values for PWT led to

heavier weights at first oestrus, an increased likelihood of conception, and a higher

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reproductive rate, compared to lower values for PWT. However, PWT did not affect age

at first oestrus or likelihood of puberty. Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight

(PWT), muscle accumulation (EMD, PEMD) and fat accumulation (FAT, PFAT) and the mean

plasma concentrations of factors involved in the regulation of tissue growth and homeostasis in

male and female lambs. GH: growth hormone (GH). IGF-I: insulin-like growth factor I. NEFA

non-esterified fatty acids.

Variable PWT EMD PEMD FAT PFAT

Male GH (r) *(0.2375) ns ns ns ns

IGF-I (r) ns ns ns **(0.2558) **(0.2868)

Ghrelin ns ns ns ns ns

Follistatin

(r)

*(0.0352) ns ns ns ns

Insulin ns ns ns ns ns

Glucose (r) *(0.2423) ns ns ns ns

Leptin ns ns ns ns ns

NEFA ns ns ns ns ns

Female GH ns ns ns ns ns

IGF-I ns ns ns ns ns

Ghrelin ns ns ns ns ns

Follistatin ns ns ns ns ns

Insulin (r) ns *(0.0584) ns ns ns

Glucose ns ns ns ns ns

Leptin ns ns ns ns ns

NEFA ns ns ns ns ns

* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns P > 0.05

Muscle tissue

Except for the weak and positive relationship between insulin and EMD in ram lambs,

EMD and PEMD were not related to the concentrations of and of the blood factors in

either sex (Table 1). With respect to reproduction in females (Table 2), a strong positive

relationship between EMD and PEMD and live weight at first oestrus was observed.

Increases in EMD and PEMD were linked to heavier weight at first oestrus and, for

EMD, an increased likelihood to attain puberty. The effect of EMD on puberty became

evident when live weight was removed from the statistical analysis.

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Adipose tissue

As for the muscle variables, FAT and PFAT were not related to the concentrations of

and of the blood factors in either sex (Table 1), with one exception – the strong positive

relationships with IGF-I values in males. For reproductive performance in females, the

only significant outcome was the positive relationship between FAT and weight at first

oestrus (Table 2).

Table 2: Correlations (r) among measures of early reproductive performance in female sheep

and post-weaning phenotypic and genotypic values for weight (PWT), muscle accumulation (EMD, PEMD) and fat accumulation (FAT, PFAT), and the mean plasma concentrations of

factors involved in the regulation of tissue growth and homeostasis. GH: growth hormone (GH).

IGF-I: insulin-like growth factor I. NEFA non-esterified fatty acids.

Variable Age 1st Oestrus

LW 1st Oestrus

Puberty Fertility Reproductive Rate

PWT ns ***(0.5662) ns * *

EMD ns ***(0.5355) * ns ns

EMD + LW ns na ns ns ns

PEMD ns *(0.2838) ns ns ns

PEMD +

PWT

ns na ns ns ns

GH ns *(–0.2755) ns ns ns

IGF-I ns ns ns ns ns

Ghrelin ns ns ns ns ns

Follistatin ns ns ns ns ns

Insulin ns *(0.1762) ns ns ns

Glucose ns ns ns ns ns

FAT ns *(0.2727) ns ns ns

PFAT ns ns ns ns ns

Leptin ns ns ns ns ns

NEFA ns ns ns ns ns

* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns P > 0.05; na not applicable

Testosterone

Testosterone concentration was strongly negatively related to values for EMD (P <

0.001; r = –0.3281), PEMD (P < 0.05; r = –0.3011) and FAT (P < 0.05; r = –0.2511).

These effects were significant whether or not live weight/PWT was included in the

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statistical analyses. Testosterone concentration was not related to changes in PWT or

PFAT.

Blood variables and reproduction

Weight at first oestrus was positively related to the circulating concentrations of insulin

and negatively related to the circulating concentrations of GH. There were no other

significant relationships between blood factors and reproductive performance (Table 2).

Discussion

The results of this study generally suggest that fast-growing lambs that gain muscle

and adipose tissue at higher rates have higher blood concentrations of factors that we

would expect to play a role in those processes (Spencer, 1985; Matzuk et al. 1995;

Florini et al., 1996; Oliver et al., 1993). Interestingly, the clearest relationships, such as

that between adipose tissue and IGF-I, were observed in males rather than females,

where there was also evidence of relationships with testosterone concentration.

Equally interesting, the significant relationships with testosterone concentration were all

negative. Overall, however, we detected very few relationships between the tissue

accumulation and any metabolic homeostatic processes, in either males or females.

With respect to reproductive performance in the females, there was a similar lack of

significant relationships, although increases in muscle accumulation, fat accumulation,

and the concentrations of GH and insulin, were all associated with greater weight at

first oestrus.

The predominance of non-significant relationships suggests that we should

reject our general hypothesis that a greater rate of prepubertal growth, specifically

accumulation of muscle or adipose tissue, would be associated with increased

concentrations of metabolites and metabolic hormones that would, in turn, improve

reproductive performance. However, we did observe several positive relationships with

reproductive success, particularly with post-weaning weight and the eye muscle

variables. This agrees with our other studies (Rosales Nieto et al., 2013a,b,c), where, it

must be noted, far more animals were used. For the present study, we needed to

restrict the number of animals so that the blood sampling protocol was technically

feasible. We also needed to select a time for the measurement of the blood factors

when the hypothetical relationships would be evident. These two compromises in the

design of the study probably limited the likelihood of detecting the critical relationships.

The issue of timing might also explain why we did not observe relationships

between the concentration of GH and phenotypic and genotypic measures of growth,

or between measures of growth and the concentrations of IGF-I and insulin, as

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established previously (Trenkle and Topel, 1978; Klindt et al., 1985; Spencer 1985;

Florini et al., 1996; Francis et al., 1998). Thus, our data do not refute the idea that

these hormones are actively involved in the regulation of the growth process (Oddy et

al., 1995; Speck et al., 1990). Indeed, we did observe (if only in females) that insulin

concentration was positively related to EMD, corroborating previous reports (review:

Spencer, 1985).

Similarly, we did not observe any relationships between muscle phenotype or

genotype and the concentrations of GH, IGF-I, leptin, glucose or follistatin, despite

strong evidence that these factors are involved in the control of muscle growth and

development (review: Oksbjerg et al., 2004; Etherton 1982; Lee and McPherron 2001;

Zeidan et al., 2005; Velloso, et al. 2008). That said, in a previous study with more

animals and a different sampling protocol, we also failed to detect relationships

between the IGF-I concentration and muscle phenotype or genotype (Rosales Nieto et

al., 2013a), yet we did detect clear positive relationships between leptin concentration

and EMD and PEMD (Rosales Nieto et al., 2013a,b). It is therefore feasible that IGF-I

is not involved in the responses to selection for increased rate of accumulation of

muscle mass. Although ghrelin itself is not involved in the process of growth and

muscle accumulation, it helps to the capture of glucose in the muscle and caused

weight gain, but did not detect any relationship with tissue accumulation (Tschop et al.,

2000; Barazzoni et al., 2007).

On the other hand, the strong relationships between measures of adipose

accumulation (FAT, PFAT) and IGF-I concentrations, observed only in males, were

contrary to the expectation that GH would suppress adipocyte growth (Etherton and

Walton, 1986; Zhao et al., 2011). Interestingly, in female sheep, both young and

mature, that had been selected for leanness or fatness, Francis et al. (1998, 1999)

reported differences in the concentrations of GH and glucose, but not IGF-I or insulin. It

is feasible that the sexes differ in these processes, perhaps due to the role played by

testosterone in males.

In the current experiment, we observed very few relationships between the

plasma concentrations of metabolites and metabolic hormones and measures of

puberty, fertility and prolificacy. Obviously, because of the protocol limitations

mentioned above, this does not imply an absence of cause-effect relationship because

there are many observations, including some from our own studies, indicating the

importance of GH, IGF-I, insulin, leptin and follistatin in the regulation of sexual

maturation and fertility (Roberts et al. 1990; Miller et al., 1995; Bourguignon 1998;

Smith et al., 2002; Jorgez et al. 2004; Chagas et al., 2007; Kimura et al. 2010; Rosales

Nieto et al. 2013a,b). For glucose and NEFA, we would have expected relationships

with reproductive performance only if the animals were not metabolically unstable

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(Canfield and Butler, 1990; Bucholtz et al. 1996; 2000) and the values that we

observed show that this was not the case.

Conclusion The single-day sampling protocol used in the present study, despite the power offered

by large numbers of serial samples from a considerable number of animals, did not

reveal convincing relationships that support physiological links between rates of

accumulation of muscle or fat and blood-borne metabolic factors that are thought to be

involved in the onset of reproduction in young sheep. There were, however, indications

that changes in the rates of growth, and accumulation of muscle and fat, affect males

and female differently. The lack of relationships between the other metabolic factors

and reproductive success suggest that we need further study with different

experimental protocols. However, studies of this type are challenging with respect to

compromises in animal number, sampling regime and timing of sampling relative to the

anticipated time of puberty, so the results are not conclusive.

Acknowledgements

The authors wish to thank to all the volunteers for their assistance in the data

collection, Mrs Margaret Blackberry (University of Western Australia) and Ms Susan

Hayward (Monash Institute of Medical Research) for their assistance with the hormone

analyses and Gavin Kearney for his advice in statistical analyses. During his doctoral

studies, Cesar Rosales Nieto was supported by CONACyT (the Mexican National

Council for Science and Technology), the Department of Agriculture and Food of

Western Australia, the Cooperative Research Centre for Sheep Industry Innovation

and the UWA School of Animal Biology.

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Chapter 4

Roles of liveweight change during mating and muscle accumulation on puberty and fertility in ewe lambs

C.A. Rosales NietoA,B,C, M.B. FergusonA,B, D, F, H. ThompsonE, J.R. BriegelB, C.A. MacleayB,

G.B. MartinC, A.N. ThompsonA,B,D,G

A CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW

2351, Australia B Department of Agriculture and Food of Western Australia, South Perth, WA 6151,

Australia C UWA Institute of Agriculture, University of Western Australia, Crawley, WA 6009,

Australia D School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150,

Australia E Moojepin MPM, Nyabing Rd Katanning, WA 6317, Australia F Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch

8024, New Zealand

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Abstract We tested the effect of liveweight change (LWC) during mating and muscle and fat

accumulation on puberty and fertility using 481 Merino ewe lambs with a range in

phenotypic values for depth of eye muscle (EMD) and fat (FAT), and a range of Australian

Sheep Breeding Values (ASBV) for post-weaning live weight (PWT), eye muscle depth

(PEMD) and fat (PFAT). From age 4 months, vasectomized rams were placed with the

ewe lambs to detect the onset of puberty. At age 8 months, the vasectomized rams were

replaced by entire rams, and the mating groups were assigned to dietary groups to

designed to target low (LOW = 40 kg, n = 244) or high (HIGH = 45 kg, n = 237) live

weights during mating. The ewe lambs weighed 39.2 ± 0.3 kg for LOW and 39.7 ± 0.3 kg

for HIGH at the start of the mating period and 39.3 ± 0.3 kg (LOW) or 45 ± 0.3 kg (HIGH)

at the end. Together, the vasectomized and entire rams detected first oestrus in 69% of

ewe lambs (average weight 37.8 ± 0.2 kg; average age 232 days, range 177-281). A

greater proportion of ewe lambs with higher values for PWT (P < 0.001) or EMD (P < 0.01)

attained puberty. HIGH females were more fertile (38% vs 7%) and had a higher

reproductive rate (P < 0.001) than LOW females. Fertility and reproductive rate were

positively associated with live weight at the start of mating (P < 0.001), regardless of level

of nutrition during mating, and with higher values for LWC, PWT and EMD (P < 0.001), and

FAT, PEMD and PFAT (P < 0.05). We conclude that increasing live weight during mating,

and the rate of muscle accumulation, will increase fertility and reproductive rate in ewes

mated at age 8 months.

Extra Keywords: Ewe lambs, nutrition, liveweight change, muscle accumulation, fertility

Introduction

Ewe lambs usually achieve puberty when they attain 50-70% of their mature live weight

(Hafez, 1952; Dýrmundsson, 1973). Therefore, important determinants of the timing of

puberty include environmental factors that influence the rate of growth, both pre- and post-

weaning (reviewed by Foster et al. 1985). Similarly, genetic factors that influence growth

will therefore affect the timing of puberty – thus Merino ewe lambs with higher breeding

values for growth attain puberty earlier and also have a higher reproductive performance

than those with lower breeding values (Rosales Nieto et al. 2013a;b). However, in ewe

lambs of similar live weight at start of mating, fertility and reproductive rate vary

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significantly within experiments and between years (Rosales Nieto et al. 2013a;b),

suggesting that other factors also determine reproductive success.

Liveweight change (LWC) during mating could contribute to this variation in

reproductive success. We observed 75% pregnancy in Merino ewe lambs that weighed 41

kg at start of mating and gained 200 g/day during mating in one study (Rosales Nieto et al.

2013a), and only 35% in another study where ewes lambs weighed 42 kg at start of mating

and gained only 50 g/day during mating (Rosales Nieto et al. 2013b). An effect of LWC

during mating on reproductive success could be explained by the ‘acute’ metabolic effect

in which short-term changes in nutrition affect ovarian function, independently of changes

in live weight, or the ‘dynamic’ effect associated with changes in live weight before

conception (reviewed by Scaramuzzi et al. 2006). However, it is not known if the ‘dynamic’

effect simply reflects weight gains during mating that lead to the ewes being heavier when

mated, or if liveweight change itself has some effect on fertility and reproductive rate in

addition to those associated with correlated changes in absolute live weight. Regardless of

the mechanism, it is expected that improving the nutrition of ewe lambs so they gain more

weight during mating will increase their fertility and reproductive rates.

In addition to live weight and LWC, differences among genotypes might also affect

the outcome of mating – for both ewe lambs and adult ewes, genotypes with higher

breeding values for muscle and fat have higher fertility and reproductive rate than

genotypes with lower values (Ferguson et al. 2007; 2010; Rosales Nieto et al. 2013a;b). In

the ewe lambs, but not adult ewes, the relationships between the reproductive

performance and body composition in ewe lambs were not evident until live weight was

excluded from the statistical analyses. Therefore, it is likely that the differences in muscle

and fat accumulation would not influence the hypothesised interaction between live weight

at mating and LWC during mating on reproductive performance. In the present study, we

tested whether improving the nutrition of ewe lambs during mating, to permit positive LWC,

would improve their fertility and reproductive rate. We also tested whether the rates of

muscle and fat accumulation are likely to be reflected in the timing of puberty and

reproductive performance.

Material and Methods

This work was done in accordance with the Australian Code of Practice for the Care and

Use of Animals for Scientific Purposes and was approved by the Animal Ethics Committee

of the Department of Agriculture and Food, Western Australia.

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Experimental location and animals

The Merino ewe lambs (n = 481) used in this study were born at ‘Moojepin’, a farm near

Katanning in Western Australia, during July-August 2010. Their mothers had been mated

to sires with a wide range in Australian Sheep Breeding Values (ASBV) for growth and for

depth of eye muscle and fat. The ASBV values delivered by MERINOSELECT were the

result of collation and analysis of individual performance values, pedigree information and

relevant environmental and management information of the animals from participating

breeders. The ewe lambs were weighed every week from 115 days before mating (Day -

115) and to 60 days after start of mating (Day 60) and these data were used to estimate

live weight at puberty, the estimated date of conception, and LWC during the experiment.

The depths of the longissimus dorsi muscle and subcutaneous fat, at a point 45 mm from

the midline over the twelfth rib, were measured using ultrasound when the ewe lambs

were on average 311 days old (range 289 to 318). The range in ewe muscle depth (EMD)

was 16-34 mm and the range in the C-site fat (FAT) was 1-8 mm. The ultrasound data

were used to generate ASBVs at post-weaning age for weight (PWT; range 0-9 kg), depth

of eye muscle (PEMD; range 0-2.6 mm) and fat (PFAT; range 0-1.2 mm) using

MERINOSELECT (Brown et al. 2007).

Animal management and feeding

Ewe lambs were maintained in a 75 ha paddock where they had ad libitum access to clean

water, oaten hay and sheep pellets. The pellets were based on barley, wheat and lupin

grains, cereal straw and hay, canola meal, minerals and vitamins, and had been

formulated to provide 11.5 MJ of metabolisable energy per kg dry matter, and 15% protein

and minerals, sufficient to meet their daily requirements for maximum growth (Macco

Feeds, Australia). A ‘Teasing Period’ was begun when 8 vasectomized Merino rams

bearing marking harnesses (MatingMark®; Hamilton, NZ) were introduced to detect the

onset of oestrus on November 26 (Day –115), when the ewe lambs were 135 days old

(range 112 to141) and weighed 25.3 ± 0.2 kg (range 15 to 40). The crayons were changed

every two weeks and crayon marks were recorded weekly to estimate the date of the first

standing oestrus. Crayon marks were scored, with Score 1 being one narrow mark on the

middle or the edge of the rump and Score 3 being one large, wide mark across the rump

(only Scores 2 or 3 were accepted as indicating standing oestrus). When the first Score 2-

3 crayon mark was recorded, the date and the age of the ewe lamb were noted. The date

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was deemed to be age at first oestrus and the closest live weight recorded to that date

was deemed to be live weight at first oestrus.

The vasectomized rams were removed on March 22 (Day 0), when lambs were on

average 244 days old (range 224 to 253 days). On Day 0, the ewe lambs were allocated

by birth type and sire to one of two dietary groups that would be fed diets designed to

achieve different target live weights during mating: a) the LOW group (target 40 kg; n =

244) received oat hay ad libitum and 300 g lupin grain per head three times per week, but

no pellets; ii) the HIGH group (target 45 kg; n = 237) received the hay and lupin grain, plus

sheep pellets ad libitum. LOW lambs weighed 39.2 ± 0.3 (range 30 to 53 kg) and HIGH

lambs weighed 39.7 ± 0.3 kg (range 30 to 53 kg) and each dietary group was then

subdivided into 4 groups of 65 on the basis of their prospective sire. Each sub-group was

placed in a separate 1 ha enclosure with access to clean water ad libitum and the

experimental diet. For the ‘Mating Period’, one experienced entire Merino ram bearing a

marking harness was introduced into each group and crayon marks were recorded weekly

and crayons changed bi-weekly. At the end of the Mating Period (Day 28), the rams were

removed and the ewe groups were combined on a common 75 ha paddock. The LOW

lambs weighed 39.3 ± 0.3 (range 29 to 54 kg) and HIGH lambs weighed 45 ± 0.3 kg

(range 33 to 58 kg). The numbers of fetuses were determined by ultrasound scanning 50

days after ram removal and the data were used to calculate fertility (percentage of

pregnant ewes per 100 ewes mated) and reproductive rate (number of fetuses in utero per

100 ewes mated).

Statistical analysis

The data were analysed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Ewe

live weight data were analyzed using the linear mixed model procedures allowing repetitive

measures (PROC MIXED) with fixed effects including dam age (years), birth type – reared

type (BTRT) and dietary group. LWC data (subdivided into the ‘Teasing’ and ‘Mating’

periods), FAT, EMD, PWT, PEMD or PFAT were each independently tested as covariates.

Identification number of the ewe within the sire (father) of the ewe lambs was used as a

random effect.

The relationships among live weight, PWT, PEMD, PFAT, EMD and FAT were

computed using PROC GLM with MANOVA option, allowing removal of major fixed effects.

Fixed effects included in the model were dam source, birth type and age at the day of the

muscle and fat scan.

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For the ‘Teasing’ and ‘Mating’ periods, LWC was determined for each lamb using a

cubic smoothing spline function with the transformation regression model procedures,

deemed appropriate when the response is nonlinear (TRANSREG). LWC data were

analyzed using the linear mixed model procedures (PROC MIXED). Fixed effects in the

model were dam age (years), BTRT and dietary group. FAT, EMD, PWT, PEMD or PFAT

were each independently tested as covariates. The sire (father) of the ewe lambs was

used as a random effect.

Live weight and age at first oestrus were analysed using mixed models (PROC

Mixed) and included: dam age (years), BTRT, live weight at the start of the ‘teasing period’

as fixed effects. LWC during the ‘Teasing Period’, FAT, EMD, PWT, PEMD or PFAT were

each independently tested as covariates. Sire (father) of the ewe lambs was used as a

random effect.

Puberty and fertility data were analyzed using the generalized linear mixed model

procedures with a binomial distribution and logit link function (PROC GLIMMIX). Fixed

effects were dam age (years), BTRT, dietary group for fertility and live weight at the start of

the ‘teasing’ period (for puberty) or the ‘mating period’ (for fertility). LWC (divided into

‘teasing period’ for puberty and ‘mating period’ for fertility), FAT, EMD, PWT, PEMD or

PFAT were each independently tested as covariates. Sire (father) of the ewe lambs was

used as a random effect. Reproductive rate data were analyzed using the generalized

linear mixed model procedures with a multinomial distribution and logit link function (PROC

GLIMMIX). The same fixed effects, covariates and random effects were used as the as for

the analysis of fertility.

All 2-way interactions among the fixed effects were included in each model and

non-significant (P > 0.05) interactions were removed from the final model. The data for

puberty, fertility and reproductive rate are presented as logit values and back-transformed

percentages.

Results

Ewe live weight and liveweight change

As shown in Figure 1, the mean (± SEM) live weight increased gradually from 25.3 ± 0.2

kg on Day –115 to 39.7 ± 0.3 kg on Day 0 when the ewe lambs were allocated to dietary

treatments. During the ‘Mating Period’ (Days 0 to 28), live weight remained stable in the

LOW group (39.3 ± 0.3 kg on Day 28) but increased to 45 ± 0.3 kg in the HIGH group.

Mean live weight differed with BTRT (P < 0.001; 35.6 ± 0.2 for single-single, 34.5 ± 1.3 for

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twin-single and 31.6 ± 0.5 for twin-twin) and dietary treatment (P < 0.01; 34.5 ± 0.3 kg for

LOW and 35.6 ± 0.3 kg for HIGH). Throughout the experiment, ewe lambs with higher

values for EMD, FAT, PWT, PEMD or PFAT (P < 0.001) were heavier than ewe lambs with

low values.

Fig. 1. Mean (± SEM) live weights of the Merino ewe lambs during the experiment. Data from single

and twin births were retained in the model and the relationship was pooled within the birth-type

classes. On Day 0 (arrow), the vasectomized rams (‘teasers’) were removed and replaced with

SIRE rams, and the animals were allocated to HIGH (○) or LOW diets (●).

During the ‘Teasing Period’, LWC was 123.8 ± 1.2 g day-1 and was not affected by

BTRT (P > 0.05). LWC was higher in ewe lambs with higher values for EMD, FAT, PWT,

PEMD or PFAT than in ewe lambs with lower values (P < 0.001). LWC during the ‘Mating

Period’ was 2.4 ± 4.2 g day-1 for the LOW group and 179.3 ± 3.8 g day-1 (P < 0.001) for the

HIGH group. LWC was higher in ewe lambs with higher values for EMD, FAT, PWT,

PEMD or PFAT than in ewe lambs with lower values (P < 0.001). BTRT had no effect on

LWC during the mating period (P > 0.05).

The correlations among live weight, PWT, EMD, PEMD, FAT and PFAT from each

dietary group are shown in Table 1. The correlations differed slightly between dietary

groups for each trait. A strong positive correlation was observed, in both dietary groups,

between LW and EMD, PWT and EMD and PEMD, EMD and FAT and PFAT and PEMD

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and PFAT. The remaining the correlations were also positive but weak for both dietary

groups (Table 1).

Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,

PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs from

LOW and HIGH nutritional groups.

Group Variable LW PWT EMD PEMD FAT PFAT

LOW LW 1 0.79 0.74 0.41 0.59 0.43

HIGH 1 0.64 0.64 0.33 0.51 0.30

LOW PWT 0.79 1 0.74 0.69 0.54 0.58

HIGH 0.64 1 0.76 0.72 0.42 0.53

LOW EMD 0.74 0.74 1 0.83 0.65 0.68

HIGH 0.64 0.76 1 0.85 0.56 0.65

LOW PEMD 0.41 0.69 0.83 1 0.54 0.77

HIGH 0.33 0.72 0.85 1 0.39 0.72

LOW FAT 0.59 0.54 0.65 0.54 1 0.84

HIGH 0.51 0.42 0.56 0.39 1 0.70

LOW PFAT 0.43 0.58 0.68 0.77 0.84 1

HIGH 0.30 0.53 0.65 0.72 0.70 1

Live weight and age at puberty

Of the 481 lambs in the flock, 332 (69%) displayed oestrus at least once during the

combined ‘Teasing’ and ‘Mating’ periods, or had cycled by the end of the Mating Period.

On average, first oestrus was observed at weight 37.8 ± 0.2 kg (range 25.2 to 51.5) and

age 233 days (range 177 to 281). The proportion of ewe lambs that attained puberty by

270 days was influenced by BTRT (P < 0.001). A greater proportion of twin-born and

single-reared ewe lambs (92%) attained puberty than females born as a single and reared

as a single (72%) or born and reared as a twin (48%). The proportion of ewe lambs that

attained puberty by Day 270 was not influenced by LWC during the ‘Teasing Period’ (P >

0.05). As values for PWT (P < 0.01) and EMD (P < 0.01) increased, it became more likely

that puberty would be reached, but this did not apply to FAT, PEMD or PFAT (P > 0.05;

Table 2).

Age and live weight at first oestrus differed with BTRT (P < 0.01). Ewe lambs born

and reared as singles were on average younger (230 days) and heavier (37.6 kg) at first

oestrus than ewe lambs born as twin and reared as single (239 days and 37.2 kg) and ewe

lambs born and reared as twin (244 days and 34.5 kg). LWC during the ‘Teasing Period’ 83

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did not influence age at first oestrus (P > 0.05; Table 2). Ewe lambs with higher values for

PWT, PFAT, PEMD, FAT or EMD were heavier at first oestrus (P < 0.001).

Ewe lambs with higher values for PWT (P < 0.001) EMD (P ≤ 0.05) and FAT (P <

0.05) were younger at first oestrus (Fig. 2). After live weight at scanning was included in

the statistical model, effects of FAT on age at first oestrus and EMD on puberty and age at

first oestrus were revealed, but not effects of PEMD and PFAT (P > 0.05; Table 2).

Table 2: Relationships among age, live weight, liveweight change (LWC; split into the teasing and

mating periods), phenotypic (Eye muscle depth [EMD] and fatness [FAT]) or ASBV (post weaning

weight [PWT], post weaning eye muscle depth [PEMD], post weaning fatness [PFAT]), and the

reproductive performance (age or live weight at first oestrus, puberty, fertility and reproductive rate)

of Merino ewe lambs mated at age 8 months.

P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05; NA Not applicable

Fertility and reproductive rate

A greater proportion of ewe lambs were pregnant at scanning in the HIGH group (38%;

90/237) than in the LOW group (7%; 18/244) (P < 0.001). Ewe lambs that were heavier at

the start of the mating period were more fertile regardless of dietary group (P < 0.001; Fig.

3).

Variable Age at 1st Oestrus

LW at 1st Oestrus Puberty Fertility Rep

Rate

Live weight (Rams in) NA NA NA *** ***

LWC (Teasing) NS NA NS NA NA

LWC (Mating) NA NA NA *** ***

PWT *** *** *** *** ***

PEMD NS *** NS ** **

PEMD (+ PWT) NS NA NS NS NS

PFAT NS *** NS * *

PFAT (+ PWT) NS NA NS NS NS

EMD * *** ** *** ***

EMD (+ Live weight at scan) NS NA NS * *

FAT * *** NS ** ***

FAT (+ Live weight at scan) NS NA NS NS *

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Fig. 2. Relationships between Australian Sheep Breeding Values for post-weaning weight (PWT; P

< 0.001) and age at first oestrus in Merino ewe lambs. The broken lines represent upper and lower

95% confidence limits.

Fig. 3. Relationships between live weight at start of mating, dietary group (LOW, grey lines; HIGH,

black lines) and fertility (P < 0.001) in Merino ewe lambs mated at 8 months of age. The broken

lines represent upper and lower 95% confidence limits.

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Fertility rate differed with dietary group (P < 0.001; 38% for HIGH vs 7% for LOW),

but not with BTRT. In those pregnant, age at conception was on average 264 d (range

256-280) for the LOW group and 260 d (range 225-280) for the HIGH group, while live

weight was 41.2 ± 1 kg for the LOW group and 43 ± 0.5 kg for the HIGH group. The

likelihood of pregnancy increased with increases in the values for LWC (P < 0.001), PWT

(P < 0.001), PEMD (P < 0.01), PFAT (P < 0.05), EMD (P < 0.001) or FAT (P < 0.01). The

effect of EMD on fertility was still evident after live weight at scanning was included in the

statistical analysis, whereas the effects of the other variables were only significant before

live weight was included (P < 0.05; Fig. 4).

Fig. 4. Fertility in Merino ewe lambs mated at 8 months of age was related Australian Sheep

Breeding Values for eye muscle depth (P < 0.05), and depended on dietary group (LOW, grey lines,

HIGH, black lines). Live weight was retained in the analysis for eye muscle depth. Data from single

and twin births were retained in the model and the relationship was pooled within the birth-type

classes. The broken lines represent upper and lower 95% confidence limits.

In the LOW group, 16/18 (89%) pregnant ewe lambs were carrying a single fetus

while 2/18 (11%) were carrying twins. In the HIGH group, 71/90 (79%) pregnant ewe

lambs were carrying a single and 19/91 (21%) were carrying twins. Reproductive rate was

influenced by dietary group (P < 0.001) but not by BTRT (P > 0.05). Reproductive rate was

positively related to live weight at start of mating, regardless of dietary group (P < 0.001).

Each extra kg from ewe lambs subjected to LOW was associated with 0.4 extra fetuses

per 100 ewes; whereas each extra kg from ewe lambs subjected to HIGH was associated

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with 3.4 extra fetuses per 100 ewes. Reproductive rate increased with increases in the

values for LWC (P < 0.001), PWT (P < 0.001), PEMD (P < 0.01), PFAT (P < 0.05), EMD (P

< 0.001) and FAT (P < 0.001). The effect of EMD and FAT on reproductive rate was still

evident after live weight at scanning was included in the statistical analysis (P < 0.05), but

the effects of PEMD, PFAT and FAT were not revealed until live weight was excluded from

the statistical analyses. An extra mm of eye muscle depth in the LOW group was

associated with 1.2 extra fetuses per 100 ewes; whereas an extra mm of eye muscle in the

HIGH group was associated with extra 5.5 extra fetuses per 100 ewes (Fig. 5).

Fig. 5. The relationships between reproductive rate and rate of muscle accumulation for the two

dietary groups (P < 0.05; LOW, grey lines; HIGH, black lines) in Merino ewe lambs mated at age 8

months. Data from single and twin births were retained in the model and the relationship was

pooled within the birth-type classes. Live weight was retained in the analysis for eye muscle depth.

The broken lines represent upper and lower 95% confidence limits. Discussion

Improved nutrition was associated with an increase in fertility and reproductive rate in

ewe lambs. The pregnancy rate in the LOW group, where live weight was maintained at

about 40 kg during mating, was lower than in the HIGH group where live weight increased

from 40 to 45 kg during mating. In the LOW group, the result did not seem to be due to

failure of ovulation because crayon marks were registered during both periods, so

impaired conception is more likely. Supporting our observations, Fletcher et al. (1970)

reported increases in pregnancy and fecundity in maiden ewes offered high levels of

nutrition during the breeding season. However, previous reports are not clear about the

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effect of an improved nutrition during mating on reproductive performance (Killeen 1967;

Gunn et al. 1992; Lassoued et al. 2004).

The positive linear effect of live weight at start of mating on the reproductive rate was

independent of the dietary group. Each extra kg from ewe lambs subjected to LOW was

associated with 0.4 extra fetuses per 100 ewes. By contrast, each extra kg from ewe

lambs subjected to HIGH was associated with 3.4 extra fetuses per 100 ewes, a value that

is consistent with our previous observation of 4.5-4.8 extra fetuses per 100 ewes (Rosales

Nieto et al. 2013a;b). Overall, it seems likely that reproductive performance may be

improved by reaching key live weights at start of mating, by better nutrition as well as by

selection of animals with higher values for growth (Rosales Nieto et al. 2013a;b).

The effect of improved nutrition was reflected in the LWC and, supporting our

hypothesis, increased LWC during mating was associated with improved reproductive

performance. The reproductive efficiency was higher with LWC above 150 g/d (HIGH) than

with LWC below 5 g day-1 (LOW). This outcome explains contrasting results in two of our

previous studies: in the first, 75% of Merino ewe lambs conceived when they were gaining

200 g day-1 during the mating period (Rosales Nieto et al. 2013a), whereas only 35%

conceived in the second experiment when the ewe lambs gained only 50 g day-1 (Rosales

Nieto et al. 2013b). Interestingly, an increase in LWC above 150 g day-1, as observed in

the HIGH group, was reflected in the total live weight, whereas an increase in LWC below

5 g day-1, as observed in the LOW group, was not. However, an increase in LWC during

the mating period has been reported elsewhere as ineffective –Mulvaney et al. (2010a;b)

did not observe any differences in pregnancy rate in Romney ewe lambs offered medium

or ad libitum diets, or gaining 100 or 200 g day-1, and Adalsteinsson (1979) did not observe

an effect of LWC during mating on fecundity in mature Icelandic ewes. The disagreements

could be due to differences in breed, age and season, or to in dietary treatments. We

nevertheless conclude that a solid gain in live weight during mating is likely to improve

pregnancy in ewe lambs. More research is needed to compare different rates of LWC

during mating and to determine whether there is a threshold in LWC for higher pregnancy

rates.

Supporting our second hypothesis, and our previous observations (Rosales Nieto et al.

2013a,b), ewe lambs with higher values for PWT grew faster, were heavier at first oestrus

and at start of the mating period, and more fertile. It is thus clear that nutritional and

selection of animals with higher values for growth, therefore both, will not only advance

puberty but also will improve the likelihood of pregnancy.

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With respect to commercial animal management, the important conclusion is that, in

ewe lambs that grow faster, through management or genetics, the first mating would be

earlier and a second mating would be feasible at 20 months of age, permitting integration

with the annual rhythm of the adult ewe flock. Such practices are also likely to reduce the

methane emissions intensity of the production system.

In addition, body composition, particularly the depths of muscle and fat, affects

reproductive performance – EMD, PEMD, FAT and PFAT were strongly related among

themselves, with age and live weight at puberty, and with fertility and reproductive rate. As

we reported previously (Rosales Nieto et al. 2013a;b), these relationships became obvious

when live weight was excluded in the statistical model, although the effect of EMD on

fertility and EMD and FAT on reproductive rate were strong enough to be evident when

live weight was retained. In the present study, high rates of muscle accumulation

increased fertility and reproductive rate, independently of weight gain during the mating

period. Interestingly, we observed that an extra mm of eye muscle depth in ewe lambs was

associated with 1.2 extra fetuses per 100 ewes in the LOW group, and an extra 5.5 extra

fetuses per 100 ewes in the HIGH group. This suggests that 1 mm more eye muscle depth

would be more beneficial than 1 kg more live weight at start of mating for the fertility in ewe

lambs. Overall, the present observations, along with those in our previous studies, strongly

support the existence of a physiological link between muscle tissue and reproductive

function in female sheep.

In the present study, rams marked most ewes during at least one of the two periods, so

reproductive failure appears to be due to failure of conception, not ovulation. Early reports

suggest that fertility remains low when live weight is below 40 kg (Coop, 1962; Killeen,

1972). Our ewe lambs weighed approximately 40 kg at start of mating, but the range within

dietary groups was 30-50 kg. In addition, recent data indicates that the ram:ewe ratio used

in the present experiment could have limited reproductive performance (B Paganoni, 2013,

unpublished data). It is clear that more research is warranted to establish the key live

weight at start of mating and the optimal of ram:ewe ratio.

Conclusion The rates of fat and muscle accumulation, and thus live weight, at start of the mating

period, strongly affect reproductive performance in ewe lambs. These attributes can be

attained by nutritional management and by selecting fast-growing animals with high values

for fat or muscle accumulation. Moreover, growth must not stop during the mating period –

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live weight gains above 150 g/d during the mating period seem to be necessary for

adequate pregnancy and reproductive rates.

Acknowledgements The authors wish to thank to Dave Thompson from Moojepin farm for allowing us to use

his animals and for his help during the experiment. During his doctoral studies, Cesar

Rosales Nieto was supported by CONACyT (the Mexican National Council for Science and

Technology), the Department of Agriculture and Food of Western Australia, the

Cooperative Research Centre for Sheep Industry Innovation, and the UWA School of

Animal Biology.

Literature cited Adalsteinsson, S (1979) The independent effects of live weight and body condition on

fecundity and productivity of Icelandic ewes. Animal Science 28, 13-23.

Brown, DJ, Huisman, AE, Swan, AA, Graser, H-U, Woolaston, RR, Ball, AJ, Atkins, KD,

Banks, RG (2007) Genetic evaluation for the Australian sheep industry. In Proceedings

of the Association for the Advancement of Animal Breeding and Genetics 17:187-194.

Coop, IE (1962) Liveweight-productivity relationships in sheep. New Zealand Journal of

Agricultural Research 5, 249-264.

Dýrmundsson, ÓR (1973) Puberty and early reproductive performance in sheep. I. Ewe

lambs. Animal Breeding Abstracts 41, 273-289.

Fletcher, I, Geytenbeek, P, Allden, W (1970) Interaction between the effects of nutrition

and season of mating on reproductive performance in crossbred ewes. Australian

Journal of Experimental Agriculture 10, 393-396.

Ferguson, MB, Adams, AN, Robertson, IRD (2007) Implications of selection for meat and

wool traits on maternal performance in Merinos. In Proceedings of the Association for

the Advancement of Animal Breeding and Genetics, 195-198.

Ferguson, MB, Young, JM, Kearney, GA, Gardner, GE, Robertson, IRD, Thompson, AN

(2010) The value of genetic fatness in Merino ewes differs with production system

and environment. Animal Production Science 50, 1011-1016.

Foster, DL, Yellon, SM, Olster, DH (1985) Internal and external determinants of the timing

of puberty in the female. Journal of Reproduction and Fertility 75, 327-344.

Gunn, RG, Milne, JA, Senior, AJ, Sibbald, AM (1992) The effect of feeding supplements in

the autumn on the reproductive performance of grazing ewes 1. Feeding fixed amounts

of supplement before and during mating. Animal Science 54, 243-248.

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Hafez, ESE (1952) Studies on the breeding season and reproduction of the ewe Part I.

The breeding season in different environments Part II. The breeding season in one

locality. The Journal of Agricultural Science 42, 189-231.

Killeen, I (1967) The effects of body weight and level of nutrition before, during, and after

joining on ewe fertility. Australian Journal of Experimental Agriculture 7, 126-136.

Killeen, I (1972) The effect of live weight on fertilization in maiden and mature Border

Leicester X Merino ewes. Proc. Aust. Soc. Anim. Prod 9, 186.

Lassoued, N, Rekik, M, Mahouachi, M, Ben Hamouda, M (2004) The effect of nutrition

prior to and during mating on ovulation rate, reproductive wastage, and lambing rate in

three sheep breeds. Small Ruminant Research 52, 117-125.

Mulvaney, FJ, Morris, ST, Kenyon, PR, West, DM, Morel, PCH (2010a) Effect of liveweight

at the start of the breeding period and liveweight gain during the breeding period and

pregnancy on reproductive performance of hoggets and the liveweight of their lambs.

New Zealand Journal of Agricultural Research 53, 355-364.

Mulvaney, FJ, Morris, ST, Kenyon, PR, Morel, PCH, West, DM (2010b) Effect of nutrition

pre-breeding and during pregnancy on breeding performance of ewe lambs. Animal

Production Science 50, 953-960.

Rosales Nieto, CA, Ferguson, MB, Macleay, CA, Briegel, JR, Martin, GB, Thompson, AN

(2013a) Selection for superior growth advances the onset of puberty and increases

reproductive performance in ewe lambs. animal 7, 990-997.

Rosales Nieto, CA, Ferguson, MB, Macleay, CA, Briegel, JR, Wood, DA, Martin, GB,

Thompson, AN (2013b) Ewe lambs with higher breeding values for growth achieve

higher reproductive performance when mated at age 8 months. Theriogenology 80,

427-435.

SAS Institute. 2008. SAS/Stat User’s guide, Version 9.2. SAS Institute Inc., Cary, NC,

USA.

Scaramuzzi, RJ, Campbell, BK, Downing, JA, Kendall, NR, Khalid, M, Muñoz-Gutiérrez, M,

Somchit, A (2006) A review of the effects of supplementary nutrition in the ewe on the

concentrations of reproductive and metabolic hormones and the mechanisms that

regulate folliculogenesis and ovulation rate. Reprod. Nutr. Dev. 46, 339-354.

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Chapter 5

Relationships among body composition, circulating concentrations of leptin and follistatin, and the onset of puberty and fertility in female sheep

C.A. Rosales Nieto,*†‡, M.B. Ferguson,*†§3, C. A. Macleay,† J.R. Briegel,† M.P.

Hedger,# G.B. Martin,‡|| A.N. Thompson,*†§2

* CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW, 2351, Australia

† Department of Agriculture and Food of Western Australia, South Perth, WA, 6151, Australia

‡ The UWA Institute of Agriculture and School of Animal Biology, University of Western Australia, Crawley,

WA, 6009, Australia

§ School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA, 6150, Australia

# Monash Institute of Medical Research, Monash University, Vic, 3168, Australia

|| Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford OX3 9DU, UK 3 Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch 8024, New

Zealand

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Abstract

The onset of puberty is linked to the attainment of critical body mass, and increases in

body mass imply accumulation of muscle and fat. Therefore, as we attempt to improve

body composition by improving the rate of muscle accumulation, we expect to affect

the onset of puberty and subsequent fertility. There is strong evidence for leptin acting

as the physiological link between adipose tissue and the reproductive axis but, for

muscle, such links have yet to be identified. One possibility is follistatin, a regulator of

muscle growth that also binds activin and has thus been implicated in the control of

reproduction. We therefore studied the relationships, during prepubertal development

in young female sheep, between circulating concentrations of follistatin and leptin and

the rates of growth and accumulation of muscle and fat, and measures of reproductive

performance. In two field experiments, we used 326 females with known phenotypic

values for depth of eye muscle (EMD) and fat (FAT) and known genotypic values for

post-weaning live mass (PWT), eye muscle depth (PEMD) and fat depth (PFAT).

Leptin concentration was positively correlated with values for EMD, PEMD, FAT, PFAT

and PWT (P < 0.001), whereas follistatin concentration was negatively correlated with

values for EMD (P < 0.001), PEMD (P < 0.01), FAT (P < 0.05) and PWT (P < 0.001).

Leptin concentration was also negatively correlated with age and live weight at first

oestrus (P < 0.05), the proportion of females that attained puberty (P ≤ 0.05), fertility (P

< 0.01) and reproductive rate (P < 0.01). By contrast, follistatin concentration was

negatively related to live weight at first oestrus (P < 0.01), fertility (P < 0.01) and

reproductive rate (P < 0.05). These observations suggest that accelerating the

accumulation of muscle and adipose tissues can advance puberty, probably due to the

stimulatory role played by leptin. By contrast, if follistatin mediates inputs from muscle

into the control of reproduction, its role appears to be inhibitory and thus would have to

be withdrawn to allow for puberty to proceed.

Keywords: Ewe lambs, follistatin, leptin, puberty, fertility

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Introduction

Puberty is usually achieved in female sheep when they attain 50-70% of their mature

body mass and recent data suggests that ewe lambs with higher genetic merit for

growth, or for the accumulation of muscle and fat, attain puberty at a younger age and

are more fertile than those with lower genetic merit (Rosales Nieto et al. 2013a,b,c).

The relationships between reproductive performance and metabolic status are

mediated by physiological signals from metabolic regulatory tissues (review: Martin et

al. 2008). For adipose tissue, the primary signal is leptin (Foster and Nagatani 1999)

but for muscle, endocrine factors associated with reproduction have not been clearly

identified. One possibility is follistatin.

Follistatin is secreted by the sheep ovary (Tisdall et al. 1992), but there is little

variation in circulating concentrations during the oestrous cycle (McFarlane et al.

2002), probably because it is highly expressed in several other tissues, particularly

muscle, where its importance for muscle growth and development has been clearly

demonstrated (Matzuk et al. 1995; Lee and McPherron 2001; Lee 2007; Gilson et al.

2009). With respect to reproduction, it seems to have no effect on the hypothalamic

GnRH secretion in sheep (Padmanabhan et al. 2002) but it appears to act at pituitary

level to inhibit FSH secretion in rodents (Ueno et al. 1987). In mice, deletion of

follistatin in adult granulosa cells reduces fertility and terminates ovarian activity

(Jorgez et al. 2004), and deletion of one isoform of follistatin reduces litter size and

leads to early cessation of reproduction (Kimura et al. 2010). Overall, it appears that

follistatin inhibits pituitary FSH synthesis and also FSH action in the ovary (Knight et al.

2012).

We tested for statistical relationships among the circulating concentrations of

leptin and follistatin, the phenotypic and genotypic values for rates of growth and

accumulation of muscle and adipose tissues, age and body mass at puberty, fertility

and reproductive rate in ewe lambs.

Material and Methods This experiment was undertaken in accordance with the Australian Code of Practice for

the Care and Use of Animals for Scientific Purposes and was approved by the Animal

Ethics Committee of the Department of Agriculture and Food, Western Australia.

Experimental location and animals

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Experiment 1 used Merino ewe lambs (n = 136) that were born in August-September

2009 on a commercial farm (‘Moojepin’). They were transported to Medina Research

Station (32.2° S, 115.8° E) where the experiment was conducted from February to

June 2010. Experiment 2 used Merino ewe lambs (n = 190) that were born in June

2010 on the research farm (‘Ridgefield’) of the University of Western Australia (32.2° S,

115.8° E). In November 2010, they were also transported to the Medina Research

Station (32.2° S, 115.8° E) for the first stage of the experiment and, in late December,

they returned to ‘Ridgefield’ where they remained until the end of the experiment. In

both experiments, the dams of the experimental animals had been sourced from two

ram breeding flocks in Western Australian and the sires had a wide range in Australian

Sheep Breeding Values (ASBV) for growth, muscle and fat. The values for ASBV

delivered by MERINOSELECT are the result of collation and and analysis of individual

performance values, pedigree information and relevant environmental and

management information of the animals from participating breeders. Data were

collected for birth date, birth weight, birth type (single or twin) and rear type to weaning

(single or twin). The ewe lambs were weighed every week and the data were used to

calculate the average daily gain (ADG) and to estimate the live body mass at puberty

and the date of conception. The depths of the longissimus dorsi muscle and

subcutaneous fat at a point 45 mm from the midline over the twelfth rib were measured

using ultrasound when the ewe lambs were aged 164 (range 134 to 176) and 251

(range 221 to 263) days for Experiment 1, and 167 (range 146 to 186) and 218 (range

198 to 228) days for Experiment 2. For both measurements for each experiment, the

range in eye muscle depth (EMD) was 20-33 mm and the range in C-site fat (FAT) was

2-8 mm. Using MERINOSELECT (Brown et al. 2007), the ultrasound data were used to

generate estimates of Australian Sheep Breeding Values at post-weaning age for

weight (PWT; range 0–9 kg), depth of eye muscle (PEMD; range 0.0–2.6 mm) and

depth of fat (PFAT; range 0.0–1.2 mm).

Animal management and feeding

The ewe lambs from Experiment 1 were initially managed as two groups in two 20 m x

60 m pens. The ewe lambs from Experiment 2 were allocated on the basis of live

weight to eight groups and housed in separate pens (6 x 14 m) at Medina research

station. The animals from both experiments had ad libitum access to water and pellets

that were introduced over a 7-day period. The pellets were based on barley, wheat and

lupin grains, cereal straw and hay, canola meal, minerals and vitamins. They were

formulated to provide 11.5 MJ of metabolisable energy per kilogram of dry matter, 15%

protein, and sufficient minerals and vitamins for maximum growth.

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On February 24 (Day - 69), when the ewe lambs from Experiment 1 were 179

days old (range 149 to 191) and weighed 37 ± 0.4 kg, four Merino wethers (rams

castrated before puberty) with harnesses (MatingMark®; Hamilton, NZ) were

introduced to detect the onset of oestrus (“pre-mating period”). The wethers had

received a 2 mL subcutaneous injection of testosterone enanthate (75 mg/mL; Ropel®,

Jurox, NSW) one week before they were placed with the ewe lambs. Every 2 weeks,

the injections were repeated and the crayons on the harnesses were changed.

For Experiment 2, the “pre-mating period” commenced on November 30 (Day -

70), when ewe lambs were on average 157 days old (range 136 to 176) and weighed

36.2 ± 0.3 kg (range 24.8 to 50.8). A vasectomized Merino ram with a marking harness

(MatingMark®; Hamilton, NZ) was introduced into each pen to detect the onset of

oestrus. On December 29 (Day -41), ewe lambs and vasectomized rams were moved

back to ‘Ridgefield’. Each pen/group was allocated to a separate 30 x 120 m plot, with

access to clean water, ad libitum oaten hay (9 Mj/kg and 9% protein) plus lupin grain

(13.5 Mj/kg and 32% protein). It was anticipated that the combination of supplement

plus dry pasture would allow the lambs to gain approximately 100 g/day.

The wethers from Experiment 1 were removed on May 4 (day 0), when the ewe

lambs were 249 (range 219 to 261) days old and weighed 41 ± 0.5 kg. The ewe lambs

received a 1 mL intramuscular injection of supplement of vitamins (Vitamin A 500,000

iu; Vitamin D3 75,000 iu; Vitamin E 50 iu/mL; Vet ADE®, Auckland, New Zealand). For

the “mating period”, they were allocated, on the basis of body mass and sire, into 8

management groups of 15 and moved into 3 m x 7 m pens where they had ad libitum

access to clean water and the sheep pellets. A single, experienced Merino ram was

introduced into each group of ewe lambs. The rams were removed on Day 47 and the

ewes remained indoors.

The vasectomized rams from Experiment 2 were removed on February 8 (Day

0) and ewe lambs were allocated on the basis of their body mass and sire into 8

groups. An experienced ram with a marking harness was introduced into each group to

begin the “mating period” when the ewe lambs were on average 226 days old (range

206 to 246) and weighed 42.4 ± 0.3 kg (range 24.3 to 56.4). The rams were removed

after 45 days.

Crayon marks on ewe rumps were recorded three times per week at Medina

and once per week at Ridgefield to estimate the date of first standing oestrus. Crayon

marks were scored (1, 2 or 3), with Score 1 being one narrow mark on the middle or

the edge of the rump and Score 3 as being a single large mark covering the rump. The

date when the first Score 2-3 crayon mark was recorded was used to estimate age at

first oestrus and the closest live weight recorded to that date was deemed to be live

weight at first oestrus. Pregnancy rate and the number of fetuses per ewe were

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confirmed by ultrasound scanning for each experiment 60 d after rams were removed.

The data from each experiment separately was used to generate values for fertility

(percentage of pregnant ewes per 100 ewes mated) and reproductive rate (number if

fetuses in utero per 100 ewes mated).

Blood sampling and immunoassay

For both experiments, 5 ml blood was sampled by jugular venipuncture on 4 occasions,

without fasting, when the ewe lambs were on average 199, 227, 248, and 269 days old

(Experiment 1) or 144, 186, 227 and 254 days old (Experiment 2). The samples were

placed immediately on ice, and then centrifuged at 2000 g for 20 min so plasma could

be harvested and stored at -20º C until hormone analysis. Plasma concentration of

total follistatin was measured in duplicate 100 μL samples by RIA using purified

heterologous bovine follistatin as standard and iodinated bovine follistatin as tracer, as

previously described (Robertson et al. 1987; Klein et al. 1991). The limit of detection

was 1.16 ng/mL and the intra- and inter-assay CVs were 7.9% and 7.8%. Plasma leptin

concentrations were determined by RIA in duplicate 100 μL aliquots, as described by

Blache et al. (2000). For Experiment 1, the limit of detection was 0.06 ng/mL and the

intra-assay CVs were 7.3% at 0.73 ng/mL, 4.4% at 0.84 ng/mL, and 2.4% at 1.61

ng/mL. For Experiment 2, the limit of detection was 0.05 ng/mL and the intra-assay

CVs were 16% at 0.47 ng/mL, 3.3% at 1.10 ng/mL, and 3.6% at 1.79 ng/mL.

Data analysis

The data were analysed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA).

Ewe body mass during the experiment was analyzed using the linear mixed model

procedures allowing repetitive measures (PROC MIXED) and included dam source,

dam age (years) and birth type as fixed effects. Follistatin or leptin concentration was

each independently tested as covariate and sire (father) of the ewe lambs was used as

a random effect.

Average daily gain (ADG) during the experiments was determined for each

lamb using a spline approach with the regression model procedures, which is

appropriate when the response is nonlinear (TRANSREG). ADG was analyzed using

the linear mixed model procedures (PROC MIXED). Fixed effects in the model were

dam source, dam age (years), birth type and age at start of the experiments. Follistatin

or leptin concentration was each independently tested as a covariate and sire (father)

of the ewe lambs was used as a random effect.

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The correlations among body mass, follistatin, leptin, PWT, PEMD, PFAT, EMD

and FAT were computed using PROC GLM with MANOVA option, allowing removal of

major fixed effects. Fixed effects included in the model were dam source, birth type and

age at the day of the muscle and fat scan.

Age and live weight at first oestrus were analysed using mixed models (PROC

Mixed) and included: dam source, dam age (years), birth type and age as fixed effects.

Concentration of follistatin or leptin was each independently tested as a covariate. Sire

(father) of the ewe lambs was used as a random effect.

Puberty and fertility data were analyzed using the generalized linear mixed

model procedures with a binomial distribution and logit link function (PROC GLIMMIX).

Fixed effects were dam source, dam age (years), birth type, age and body mass at the

sampling date. Concentration of follistatin or leptin was each independently tested as a

covariate. Sire (father) of the ewe lambs was used as a random effect. Reproductive

rate data was analyzed using the generalized linear mixed model procedures with a

multinomial distribution and logit link function (PROC GLIMMIX). The same fixed

effects, covariates and random effects were used as for the fertility analysis.

Average body mass, PWT, EMD, PEMD, FAT and PFAT were analysed using

mixed models (PROC MIXED) and included the fixed effects: dam source, dam age

(years), and birth-reared type. Average hormone concentration (leptin or total follistatin)

was each independently tested as a covariate. Sire (father) of the ewe lambs was used

as a random effect.

Hormone concentration (follistatin, leptin) was analysed using mixed models

(PROC MIXED) allowing for repeated-measures, and included the fixed effects: dam

source, dam age (years), birth-reared type and age and body mass at start of teasing.

FAT, EMD, PWT, PEMD or PFAT were each independently tested as a covariate. Sire

(father) of the ewe lambs was used as a random effect. Mean hormone concentration

was analyzed using analysis of variance model procedures, where Factor A was

hormone concentration and Factor B was date at sampling (PROC ANOVA).

All 2-way interactions among the fixed effects were included in each model and

non-significant (P > 0.05) interactions were removed from the final model. The data for

puberty, fertility and reproductive rate are presented as logit values and back-

transformed percentages.

Results

Body mass, leptin and follistatin

In Experiment 1, mean body mass increased from around 37 kg on Day –75 to around

54 kg on Day 57 (Figure 1A), with an ADG of 144 ± 2.4 g. There was a clear set-back

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in growth between Days -20 and +20, around the time of introduction of fertile males.

Mean leptin concentration increased from 1.3 ng mL-1 on Day –50 to 1.7 ng mL-1 on

Day +20, (P < 0.001; Figure 1B), although the progressive increase was also

interrupted around Day 0 at the time of the arrest in weight gain (upper panel). By

contrast, total follistatin concentration decreased gradually from 3.1 ng mL-1 on Day –

50 to 2.7 ng mL-1 on Day 0 (P < 0.001), after which it did not change (Figure 1B).

In Experiment 2, body mass increased from 37 kg on Day -75 to 48 kg on Day

59 (Figure 1C). On several occasions, growth was briefly negative (eg, Days –50,–12,

–8, +24) so the overall ADG (69 ± 4.7 g) was about half that observed in Experiment 1.

In Experiment 2, Leptin concentrations remained around 2.0 to 2.2 ng mL-1 except on

Day 0 (Figure 1D) when there was a temporary but major decline (P < 0.001) that

appeared to be associated with a brief arrest in growth. By contrast, total follistatin

concentration decreased markedly from 5 ng mL-1 on Day -80 to around 3.5 ng mL-1 on

Day 0 (P < 0.001; Figure 1D), before rising again when growth appeared to stop

(Figure 1C).

Figure 1. Changes in body mass in Experiment 1 (A) and Experiment 2 (C) and circulating

concentrations of follistatin (○) and leptin (●) in the Merino ewe lambs in Experiment 1 (B) and

Experiment 2 (D). Day 0 is the day fertile Merino rams were introduced. Values are mean ±

SEM.

Leptin concentration was strongly positively correlated with body mass in both

experiments (P < 0.001; Figure 2A, B). By contrast, for follistatin concentration, the

C

-80 -60 -40 -20 0 20 40 601.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Days of the experiment

D

Pre-mating period Mating period

35

40

45

50

55A

-80 -60 -40 -20 0 20 40 602.0

3.0

4.0

5.0

B5.5

2.5

3.5

4.5

Days of the experiment

Pre-mating period Mating period

Bod

y m

ass

(Kg)

Folli

stat

in(n

g/m

L-1 ) Leptin

(ng/mL

-1)

99

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relationship was significant but weak in Experiment 1 (P < 0.01; Figure 2A) and not

significant in Experiment 2 (Figure 2B).

Figure 2. Correlation between body mass and circulating concentrations of follistatin (○ grey

lines) and leptin (● black lines) in Merino ewe lambs in Experiment 1 (A; P < 0.01 for follistatin

and P < 0.001 for leptin) and Experiment 2 (B; P > 0.05 for follistatin and P < 0.001 for leptin).

Growth, muscle and fat

The correlations among live weight, PWT, EMD, PEMD, FAT and PFAT are shown in

Table 1 for both experiments. In both experiments, strong positive relationships were

observed between EMD and live weight and PWT, live weight and FAT, and PEMD

and PFAT; the remaining relationships were also positive but relatively weak (Table 1).

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Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,

PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs from

Experiments 1 and 2.

Experiment Variable LW PWT EMD PEMD FAT PFAT

1 LW

1 0.68 0.64 0.28 0.56 0.29

2 1 0.79 0.70 0.24 0.52 0.26

1 PWT

0.68 1 0.53 0.40 0.39 0.34

2 0.79 1 0.63 0.29 0.33 0.11

1 EMD

0.64 0.53 1 0.41 0.59 0.34

2 0.70 0.63 1 0.76 0.54 0.52

1 PEMD

0.28 0.40 0.41 1 0.22 0.76

2 0.24 0.29 0.76 1 0.40 0.67

1 FAT

0.56 0.39 0.59 0.22 1 0.26

2 0.52 0.33 0.54 0.40 1 0.71

1 PFAT

0.29 0.34 0.34 0.76 0.26 1

2 0.26 0.11 0.52 0.67 0.71 1

Leptin concentration was strongly and favourable related to PWT, EMD, FAT

and PFAT in both experiments. The correlation with PEMD was also significant in both

experiments, but strong and favourable in Experiment 2 and relatively weak and

favourable in Experiment 1. By contrast, where the relationships with follistatin

concentration were significant, they were all negative. In Experiment 1, the

relationships were strong for PWT, EMD, PEMD and FAT but, in Experiment 2, there

were only two significant correlations, with EMD and PEMD, and both were relatively

weak and favourable (Table 2).

Potential effects of muscle accumulation on leptin and follistatin concentrations

are of particular interest, so the relationships with EMD are explored in Figure 3. For

leptin, the relationships are relatively robust in both experiments whereas, for follistatin,

the relationships are also significant but explain only 2-10% of the variation.

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Table 2. Correlations (r) among post-weaning phenotypic and genotypic values for weight

(PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) and the circulating

concentrations of leptin and total follistatin in Merino ewe lambs. Information has been provided

for analyses with or without live weight (LW, PWT) included in the statistical model.

Experiment 1 Experiment 2

Variable Leptin Follistatin Leptin Follistatin

PWT (r) *** (0.46) *** (-0.28) *** (0.27) NS

EMD (r) *** (0.47) *** (-0.32) *** (0.55) * (-0.15)

EMD + LW * ** *** *

PEMD (r) * (0.19) ** (-0.27) *** (0.53) ** (-0.18)

PEMD + PWT NS * *** **

FAT (r) *** (0.41) ** (-0.23) *** (0.47) NS

FAT + LW *** NS *** NS

PFAT (r) *** (0.26) NS *** (0.45) NS

PFAT + LW NS NS *** NS

Puberty

As shown in Table 3, leptin concentration was positively associated with the proportion

of ewe lambs than attained puberty for Experiment 2 (P ≤ 0.05), but the relationship

was not significant for Experiment 1 (P = 0.08). Age at first oestrus was weakly

negatively correlated with leptin concentration in Experiment 1 (P < 0.01), but the

association was not significant in Experiment 2 (P > 0.05). Live weight at first oestrus

was weakly positively correlated with leptin concentration for Experiment 2 (P < 0.05),

but not Experiment 1. These effects of leptin concentration only were evident before

live weight was included in the statistical analysis (Table 3). Follistatin concentration

was not related to the proportion of ewe lambs entering puberty or to age at first

oestrus. There was a weak and significant negative relationship with live weight at first

oestrus, but only in Experiment 1 (Table 3).

Fertility and reproductive rate after puberty

As shown in Table 2, for Experiment 1, leptin concentration was positively correlated

with the proportion of ewe lambs that became pregnant (P < 0.01) and with

reproductive rate (P < 0.01). By contrast, follistatin concentration was negatively

correlated with the proportion of ewe lambs that became pregnant (P < 0.01) and with

reproductive rate (P < 0.05). The effect of leptin on fertility and reproductive rate was

evident whether live weight was included or not in the statistical analyses, but the

effects of follistatin on fertility and reproductive rate were only significant when live

weight was not included.

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The strong but contrasting relationships observed in Experiment 1 between

fertility and leptin, and between fertility and follistatin, are illustrated in Figure 4. For

Experiment 2, neither leptin nor follistatin concentration were related to the proportion

of ewe lambs that became pregnant or with reproductive rate (Table 3). Figure 3. Correlation analysis for the effect of depth of eye muscle (EMD) on the concentrations of

follistatin (○ grey lines) and leptin (● black lines) in Merino ewe lambs from Experiment 1 (A; P <

0.05 for leptin and P < 0.01 for follistatin) and Experiment 2 (B; P < 0.001 for leptin and P < 0.05

for follistatin).

Follistatin (ng/mL-1) Leptin (ng/mL-1)

Depth of Eye muscle (EMD; mm)

By = -0.0924x + 6.2031

R² = 0.0226

y = 0.0571x + 0.3721R² = 0.2484

0.8

1.2

1.6

2

2.4

2.8

1

2

3

4

5

6

7

8

17 21 25 29 33

Ay = -0.1633x + 7.3099

R² = 0.0957

0.8

1.2

1.6

2

2.4

2.8

1

2

3

4

5

6

7

8 y = 0.0458x + 0.2496R² = 0.1967

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Table 3. The correlations (r) between the hormone concentrations (follistatin and leptin) and the

advent of puberty, the age and live weight at first oestrus, and reproductive performance

(fertility, reproductive rate) in Merino ewe lambs mated at 8-9 months of age. Information has

been provided for analyses with or without live weight (LW) included in the statistical model

except for fertility where both outcomes are provided.

Experiment 1 Experiment 2

Variable Leptin Follistatin Leptin Follistatin

Puberty (%) NS NS * NS

Age at first oestrus (days) (r) ** (-0.21) NS NS NS

Age at first oestrus + LW NS NS NS NS

LW at first oestrus (kg) (r) NS * (-0.17) * (0.19) NS

Fertility (%) ** ** NS NS

Fertility + LW * NS NS NS

Reproductive Rate (%) ** * NS NS

Reproductive rate + LW * NS NS NS

Discussion

In these field studies with Merino ewe lambs, circulating concentrations of leptin

increased progressively as the animals grew and puberty approached, consistent with

previous reports (Foster and Nagatani 1999). The most consistent relationships, across

both experiments, showed the dependence of circulating leptin concentrations on the

rate of growth and the rates of muscle and fat accumulation. Thereafter, leptin

concentration was itself positively related to age and live weight at first oestrus, as well

as to the success of puberty and fertility, and reproductive rate, observations that are

consistent with previous studies (reviewed by Smith et al. 2002). Overall, our

observations support the idea that increases in the rates of accumulation of adipose

tissue, perhaps acting through the leptin that it produces, will inform to the brain

centres that control reproduction about the body composition of the animal and

advance the onset of puberty.

Follistatin concentrations, by contrast, decreased as the animals grew and the

amount of muscle increased. Interestingly, the concentration of total follistatin was

similarly low at the onsets of puberty and conception in both Experiments. Moreover,

whenever there were significant relationships with measures of reproductive function,

they were negative. These observations are all consistent with the suggestion that, in

young female sheep, follistatin delays puberty and reduces fertility and reproductive

rate. If circulating follistatin acts as a physiological signal between muscle tissue and

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the reproductive axis, it appears to be an inhibitory factor that needs to be reduced

before reproduction can proceed.

Figure 4. Effect of leptin (black line) and follistatin (grey line) concentration on fertility in the

Merino ewe lambs from Experiment 1. The broken lines represent upper and lower 95%

confidence limits (both relationships: P < 0.01).

There were inconsistencies among the observations from these two field

studies. For example, the dependency of leptin concentration on the rates of growth

and the rates of muscle and fat accumulation significant in both experiments, but

relationships were stronger in Experiment 2 than in Experiment 1, particularly for

phenotypic and genotypic measures of muscle accumulation (EMD, PEMD). For

follistatin concentration, on the other hand, relationships with the rates of growth and of

muscle and fat accumulation were weaker for Experiment 1 than for Experiment 2. Ewe

lambs from Experiment 2 were heavier and had higher circulating concentrations of

both hormones than the ewe lambs from Experiment 1 but the average growth rate was

lower for Experiment 2 than for Experiment 1. Our interpretation is differences between

the experiments is largely explained by variation in growth rate during the mating

period, a variable that is difficult to control with field studies in an extensive

management system. It is clear that, under these conditions, external factors affected

the secretion of leptin and follistatin, and the reproductive performance of ewe lambs.

The relationships described above were revealed because we used large numbers of

animals.

Interestingly, we observed significant relationships between muscle

accumulation and leptin concentration and between muscle accumulation and

reproductive performance. Leptin is thought to be produced primarily by adipose tissue,

1 1.5 2 2.5 3 3.5

0

20

40

60

80

100

1 2 3 4 5 6 7 8

Fert

ility

(%)

Follistatin (ng/mL-1)

Leptin (ng/mL-1)

105

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but the large variation in circulating leptin concentrations at similar levels of adiposity

implies roles for factors other than fat mass (Flier 1997). Our observations suggest that

intramuscular adipose tissue might also be a biologically significant source of leptin, a

concept supported by other findings: the leptin gene is expressed in muscle; leptin

induces muscular hypertrophy and regulates energy expenditure and fat oxidation in

muscle; and, as muscle mass increases, the concentration of intramuscular fat

increases (Gong et al. 1997; Muoio et al. 1997; Wang et al. 1998; Zeidan et al. 2005;

Zhong et al. 2011). It is therefore possible that the leptin produced in muscle,

particularly in intramuscular fat, might work together in parallel with that from adipose

tissue to inform the brain about metabolic reserves.

We hypothesized that follistain concentration would be high during muscle

growth and development, and therefore higher in ewe lambs selected for rapid muscle

accumulation. We have rejected these hypotheses because all of the relationships that

we observed were negative. Our observations were made under field conditions so is

possible that day-to-day variations in food intake and weight gain affected follistatin

secretion (review: Silanikove 2000). Indeed, Phillips et al. (1998) reported that

restriction or reduced food intake affected circulating concentrations of follistatin in

Romney ewe lambs. On the other hand, we also need to explore the relationship

between myostatin and follistatin. Myostatin is an important inhibitory regulator of

muscle development (McPherron et al. 1997; Thomas et al. 2000; Lee and McPherron

2001) and a mutation in the myostatin gene and increases in the production of growth

factors, including follistatin, are responsible for enhanced muscle development in

sheep (Clop et al. 2006). However, in animals selected for accelerated muscle

accumulation, but less specialized for meat (eg Merino), the ratio of follistatin to

myostatin is probably more important in determining muscle mass (McPherron et al.

1997). To date, there have been no studies of the processes that underpin muscle

accumulation in Merino sheep that have been selected for rapid muscle accumulation.

With respect to reproduction, the decline in total follistatin concentration at the

approach of puberty and conception reflects previous observations (Foster et al. 2000;

McFarlane et al. 2002). Among the variety of effects that follistatin has been reported to

have on the reproductive axis, our observations are consistent with its role in blocking

the activins – at pituitary level, reducing FSH synthesis and at ovarian level reducing

the actions of FSH on granulosa cells. Under these circumstances, withdrawal of

follistatin as mature body mass is achieved would aid the progress of puberty and the

maximization of fecundity.

In conclusion, selection for rapid accumulation of muscle or fat will advance

puberty and improve reproductive performance in sheep, apparently because the

changes in tissue accumulation affect the circulating concentrations of leptin and

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follistatin. These hypotheses have been generated from correlations with large

numbers of animals in field studies, and now need to be tested in intervention

experiments.

Acknowledgements The authors wish to thank to Mrs Margaret Blackberry (University of Western Australia)

and Ms Susan Hayward (Monash Institute of Medical Research) for their assistance

with the hormone analyses and Gavin Kearney for his advice in statistical analyses.

During his doctoral studies, Cesar Rosales Nieto was supported by CONACyT (the

Mexican National Council for Science and Technology), the Department of Agriculture

and Food of Western Australia, the Cooperative Research Centre for Sheep Industry

Innovation and the UWA School of Animal Biology.

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General Discussion

The aim of the studies in this thesis was to test the general hypothesis that increases in

the rate of growth and, specifically, the rates accumulation of muscle and adipose

tissues, during pre-pubertal life, will advance puberty and improve subsequent

reproductive success. As a secondary consideration, it was expected that this

connection between accelerated growth and early puberty would be mediated by

changes in the circulating concentrations of the hormones produced by the key tissues,

follistatin from muscle and leptin from adipose tissue.

The general hypothesis was supported by observations in several experiments

involving over 840 Merino ewe lambs over two seasons. We found that ewe lambs with

higher phenotypic or genotypic (breeding) values for growth were more likely to attain

puberty at a younger age, and then conceive, than their counterparts with low potential

for growth. This was strongly linked to the fact that lambs selected for rapid growth

reached the ‘critical weight’ for puberty earlier in life. The relationships between

accelerated growth and puberty were observed initially in the study described in

Chapter 1 and later corroborated by the studies described in Chapters 2 and 4. These

findings extend on those by Hawker and Kennedy (1978) and Alkass, et al. (1994) who

showed that ewes that grew faster reached puberty at a younger age. In addition to

puberty, we found that the fertility rates could be improved with higher genetic values

for growth (Chapters 1, 2 and 4), supporting previous reports of a positive genetic

correlation between weaning weight, rapid growth and reproductive performance in

young female sheep (Barlow and Hodges 1976; Adalsteinsson 1979; Gaskins, et al.

2005). Ewe lambs with greater potential for growth were heavier at the start of mating

and grew faster during mating, and both of these factors were associated with an

increase in fertility.

Most of the ewe lambs in the experiments described here achieved puberty

when they had achieved 50-70% of estimated mature body weight, a threshold for

spontaneous puberty in sheep that was established long ago (Hafez, 1952;

Dýrmundsson, 1973). Our data reinforce the observation that fast growth does not limit

the ability of ewe lambs to attain the critical percentage of mature live weight,

suggesting that there is significant scope for selecting animals for higher growth

potential with positive outcomes for reproductive efficiency as well as meat production.

This strategy should enable the mating of Merino ewe lambs at a younger age and with

better fertility. Reaching puberty earlier also increases the likelihood that, when these

ewe lambs mate for a second time at 20 months of age, their reproductive cycle can be

integrated with that of the adult ewe flock.

111

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In Chapters 1, 2 and 4, we confirmed that live weight at the start of mating plays

an important role in determining fertility and reproductive rate, expanding upon

previous observations in 18-month-old maiden ewes by Kleemann and Walker (2005).

We found a linear response between pregnancy rate and live weight at the start of

mating, over the ranges of 30 to 45 kg (Chapter 1) and 40 to 55 kg (Chapter 2). There

was also a linear response in reproductive rate to increases in live weight at start of

mating. Each extra kg from ewe lambs at the start of mating was associated with 4.5

(Chapter 1) or 4.8 (Chapter 2) extra fetuses per 100 ewes. The range of 40-45 kg

seems narrow but it covers major differences in performance for Merino ewe lambs. In

Chapter 4, for example, we found that the pregnancy rate was lower in the group that

started with a body weight of 40 kg and only maintained it during mating, than in the

group that started at 40 but grew to 45 kg during mating. Each extra kg in the lambs

that maintained low weight was associated with 0.4 extra fetuses per 100 ewes;

whereas each extra kg in ewe lambs gained weight was associated with 3.4 extra

fetuses per 100 ewes. Moreover, from comparison with other studies in our laboratory

(Ferguson, et al. 2011), it seems that improving live weight at the start of mating will

provide a greater benefit for ewe lambs (3.4-4.8 extra fetuses per 100 ewe lambs) than

for mature ewes (1.7-2.4 extra fetuses per 100 mature ewes). This gain in reproductive

performance in ewe lambs, by ensuring that they reach the key live weight at start of

mating, can be achieved through nutritional management and probably also through

genetic selection for fast growth.

Comparison of the experiments in this thesis suggests that conception rate is

affected by live weight change (LWC) during mating. In the study described in Chapter

1, 75% of Merino ewe lambs conceived with an LWC of 200 g/day during the mating

period whereas, in Chapter 2, only 35% conceived with an LWC of 50 g/day during

mating. This is supported by the study described in Chapter 4 for the LWC range of 5

to 150 g/d. The literature is controversial for this topic, but our data support the view

that a positive LWC above 150 g/d during the mating period is necessary for

maximizing pregnancy success.

An important aspect of the studies in this thesis was the dissection of effects

based on total body mass into effects that can be specifically attributed to major,

physiologically active tissues. Compared to lambs with low phenotypic and genotypic

values for fat and muscle, lambs with high values were younger and heavier at first

oestrus, conceived more readily, and had a higher reproductive rate. These

observations apply regardless of whether the ewe lambs were fed ad libitum during

mating (Chapter 1) or fed restricted diets during mating (Chapter 2). In many cases,

these relationships did not become evident until live weight was removed from the

statistical model, thus allowing us to focus on biologically active constituents of body

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mass. In Chapter 4, on the other hand, the relationship with adiposity potential and age

at first oestrus, and the relationship between muscling potential and fertility and

reproductive rate, were still evident when live weight was retained in the statistical

model. These observations extend on those for mature Merino ewes by Ferguson, et

al. (2007), who reported a positive correlation between fecundity and the rate of muscle

accumulation. We therefore support the well-established concept that adipose tissue is

an important determinant of puberty and add a novel dimension of great interest – a

potential link between muscle accumulation and reproductive function.

The final step in our attempts to understand the relationships between growth or

body composition and puberty and reproductive performance was an assessment of

potential physiological links that might be involved. We focused on metabolic factors

that were likely to be affected by rates of growth and muscle and fat accumulation, and

that have been implicated in the regulation of sexual maturation and reproductive

performance (review: Martin, et al. 2008). The primary candidate from adipose tissue is

leptin, a potent regulator of appetite and metabolism that has shown to directly affect

the brain processes that control the initiation of puberty and modulate fertility (reviews:

Foster and Nagatani 1999; Blache, et al. 2000; Smith, et al. 2002). With respect to

muscle, however, where endocrine factors associated with reproductive performance

have not been clearly identified, the literature guided us to IGF-I and follistatin.

IGF-I is important in skeletal muscle growth and development (review:

Oksbjerg, et al. 2004; Duan, et al. 2010) and increases on IGF-I concentration during

prepubertal growth have been associated with the onset of puberty in female sheep

(Roberts, et al. 1990). Follistatin is secreted by the ovary, but there is little variation in

circulating concentrations during the oestrous cycle so other tissues, such as muscle,

have become the focus of attention (McFarlane, et al. 2002). In mice, follistatin is

important for muscle growth and development, and overexpression increases muscle

weight and mass (Matzuk, et al. 1995; Lee and McPherron 2001; Lee 2007; Gilson, et

al. 2009). The importance of follistatin in reproduction seems to be clear for mice: first,

deletion of follistatin in adult granulosa cells reduces fertility and terminates ovarian

activity (Jorgez, et al. 2004); second, deletion of one isoform of follistatin reduces litter

size and leads to early cessation of reproduction (Kimura, et al. 2010).

In Chapter 1, we found that the circulating concentrations of leptin and IGF-I

increased progressively as live weight increased and as puberty approached,

confirming previous reports (Roberts, et al. 1990; Foster and Nagatani 1999). This

suggests that these two metabolic hormones inform the central nervous system of the

metabolic status of the body, perhaps specifically the accumulation of fat (leptin) and

muscle (IGF-I), and thus lead to the triggering of puberty. After puberty, the

concentration of leptin was associated with fertility and reproductive rate, consistent

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with earlier observations (reviewed by Smith, et al. 2002). In Chapter 2, we observed

an increase in leptin concentration as puberty approached, but the relationship

between leptin and puberty or fertility was not statistically significant. The concentration

of ghrelin was also measured in this chapter, but it was not associated with any aspect

of reproductive rate. However, in Chapter 3, we did not detect relationships between

the concentrations of metabolic factors (GH, IGF-I, insulin, glucose, NEFA, leptin,

ghrelin, follistatin) and the rates of growth, muscle or fat accumulation. Furthermore, in

ewe lambs, the concentration of these metabolites and metabolic hormones did not

seem to be linked to reproductive performance. However, studies of this type are

challenging with respect to compromises in animal number, sampling regime and

timing of sampling relative to the anticipated time of puberty, so the results are not

conclusive.

Finally, in Chapter 5, we attempted to separate the potential effects of adipose

and muscle tissues. We observed that the most consistent relationships showed the

dependence of circulating leptin concentrations on the rate of growth and the rates of

muscle and fat accumulation. Thereafter, leptin concentration was itself positively

related to age and live weight at first oestrus, as well as to the success of puberty and

fertility, and reproductive rate, observations that are consistent with previous studies

(review: Foster and Nagatani, 1999; Smith, et al. 2002). On the other hand, we found

that follistatin concentrations decreased as the animals grew and the amount of muscle

increased. Notably, the concentration of total follistatin was similar at the onsets of

puberty and conception. Moreover, whenever there were significant relationships

between follistatin concentrations and measures of reproductive function, they were

negative. Taken together, these observations are consistent with the suggestion that, in

young female sheep, follistatin delays puberty and reduces fertility and reproductive

rate. Our observations support the idea that increases in the rates of accumulation of

adipose tissue, perhaps acting through the leptin that it produces, will inform to the

brain centres that control reproduction about the body composition of the animal and

advance the onset of puberty. However, by contrast, if circulating follistatin is acting as

a mechanistic link between muscle tissue and the reproductive axis, it appears to be an

inhibitor that needs to be removed before reproduction can proceed.

Another possible explanation of the relationship between muscle accumulation

and reproduction could be leptin. The dogma states that leptin is produced mostly by

the major white adipose deposits and observations in this thesis support that view –

leptin concentrations increased in parallel with adipose stores and were strongly

correlated with live weight and body composition (Chapters 1 and 2). However, we also

observed positive correlations between leptin concentrations and the two measures of

muscle development, PEMD and EMD. Indeed, there is large variation in circulating

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leptin concentrations at similar levels of adiposity so factors other than simple fat mass

must be involved (Flier, 1997). Observations in Chapters 1, 2 and 5 are consistent with

the possibility that intramuscular adipose tissue is a biologically significant source of

leptin. This concept is supported by other findings: the leptin gene is expressed in

muscle; leptin induces muscular hypertrophy and regulates energy expenditure and fat

oxidation in muscle; and, as muscle mass increases, the amount of intramuscular fat

increases (Gong, et al. 1997; Muoio, et al. 1997; Wang, et al. 1998; Zeidan, et al. 2005;

Zhong, et al. 2011). It is therefore possible that the leptin produced in intramuscular fat

works together in parallel with that from adipose tissue to inform the brain about

metabolic reserves.

Conclusion The basic process underlying these studies was a search for statistical relationships to

support a priori hypotheses that were based on known physiological processes in the

control of the reproductive system, especially those controlling the timing of puberty

and early reproductive performance. Overwhelmingly, the observations support the

general hypothesis that increases in the rate of growth and, specifically, the rates

accumulation of muscle and adipose tissues, during pre-pubertal life, will advance

puberty and improve subsequent reproductive success. With respect to the

physiological connections between accelerated growth and early puberty, the roles of

the circulating concentrations of the hormones produced by the key tissues are less

clear. There are very positive signs, particularly for leptin and follistatin, but conclusive

evidence awaits experiments with alternative sampling protocols and intervention

treatments.

Finally, the studies in this thesis provide a very solid foundation on which to

develop industry strategies for genetic improvement of body composition, particularly

with a view to more rapid muscle (carcase) development. The advantages of such

strategies for the meat industry are obvious but it seems very likely that increasing the

rate of muscle accumulation will also advance puberty and improve the overall

reproduction rate of the flock. The goal of strong reproductive performance in the first

year of life is within reach, as are a major step-up in the lifetime reproductive

performance of each ewe, simply through an extra year of lamb production. Finally,

young ewes will not waste a year of productive life whilst only producing methane, so

early fertility will contribute to a major reduction in emissions intensity. We therefore

have three reasons to continue to pursue this line of investigation.

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