Factors affecting differences between birth weight of littermates (BWTD) and the effects of BWTD on lamb performance.

The objectives of this study were to determine factors affecting lamb birth weight (BWT) and differences between BWT of the largest and smallest littermates (BWTD) and to assess the relative importance of BWT and BWTD on lamb survival and growth to weaning. Records from twin (n = 5369) or triplet (n = 1664) litters born on pasture were utilised. Breeds included Coopworth, Romney, Perendale, composite and Texel. Ewe and sire breed, age and weight of the dam, sex, and number of lambs born (NLB) affected BWT. Neither embryo loss nor ovulation pattern (i.e. all ova released from one ovary or some from each) were associated with BWT. Dam weight and NLB affected BWTD but dam age, sire or dam breed did not. Loss of an embryo reduced BWTD, and BWTD was lower when the ewe ovulated from both ovaries versus only one ovary for twins, but not triplets. Whereas BWT was moderately heritable (h2 = 0.20), BWTD was not heritable (h2 = 0.003). Lamb survival was positively associated with BWT. Lambs with BWTD > 1.3 kg were less likely to survive (73.3%) than those from litters of smaller BWTD (range 82.8-85.7% survival). Growth rate of twin and triplet lambs was positively associated with BWT. Surprisingly, lambs from litters with moderately high BWTD had slightly greater (approximately 3%) growth rate than those from lambs of low to intermediate BWTD. Thus, while large BWTD were negatively associated with survival, BWT itself, which was moderately heritable, appeared to be a stronger driver of lamb survival and average daily gain.

Differential expression of CART in ewes with differing ovulation rates.

We hypothesised that cocaine- and amphetamine-regulated transcript (CARTPT) would be differentially expressed in ewes with differing ovulation rates. Expression of mRNA for CARTPT, as well as LHCGR, FSHR, CYP19A1 and CYP17A1 was determined in antral follicles ≥1 mm in diameter collected during the follicular phase in ewes heterozygous for the Booroola and Inverdale genes (I+B+; average ovulation rate 4) and ++ contemporaries (++; average ovulation rate 1.8). In ++ ewes (n = 6), CARTPT was expressed in small follicles (1 to <3 mm diameter), where 18.8 ± 2.5% follicles expressed CARTPT CART peptide was also detected in follicular fluid of some follicles of ++ ewes. In I+B+ ewes, 5/6 ewes did not have any follicles that expressed CARTPT, and no CART peptide was detected in any follicle examined. Expression pattern of CYP19A1 differed between I+B+ and ++ ewes with an increased percentage of small and medium follicles (3 to <4.5 mm diameter) but decreased percentage of large follicles (≥4.5 mm diameter) expressing CYP19A1 in the I+B+ ewes. Many of the large follicles from the I+B+ ewes appeared non-functional and expression of LHCGR, FSHR, CYP17A1 and CYP19A1 was less than that observed in ++ ewes. Expression of FSHR and CYP17A1 was not different between groups in small and medium follicles, but LHCGR expression was approximately double in I+B+ ewes compared to that in ++ ewes. Thus, ewes with high ovulation rates had a distinct pattern of expression of CARTPT mRNA and protein compared to ewes with normal ovulation rates, providing evidence for CART being important in the regulation of ovulation rate.

Reduced ovulation rate, failure to be mated and fertilization failure/embryo loss are the underlying causes of poor reproductive performance in juvenile ewes.

A ewe that is mated as a juvenile (producing a lamb at 1 year of age) will produce an average of only 0.6 lambs to weaning, compared to an average of 1.2 lambs in adult ewes. Understanding the underlying causes of this low reproductive efficiency and designing methods to improve or mitigate these effects could potentially increase adoption of mating juvenile ewes. In Experiment 1, 2 Cohorts of ewes, born a year apart, were mated in order to lamb at 1 and 2 years of age and the performance of the ewes at each age was compared. Onset of puberty, mating by the fertile ram, ovulation rate, early pregnancy (day 30-35) litter size, number of lambs born and number of lambs weaned were measured. In juvenile ewes, by day 35 of pregnancy, 43% of ova had failed to become a viable embryo and this early loss was the largest contributor to the poor reproductive performance observed. Compared with young adult ewes, ovulation rate was lower (p<0.001), fewer ova were exposed to sperm (p<0.001) and fertilization failure/embryo loss was increased (p<0.001) in juveniles. In Experiment 2, the early pregnancy litter size of juveniles was shown to be greater (p<0.001) in those ewes with a greater ovulation rate (p<0.001). Attaining puberty prior to introduction of the fertile ram was associated with an increased pregnancy rate (p<0.001). In juvenile ewes, failure to mate with the ram, lower ovulation rate and increased fertilisation failure/embryo loss underlie their poor reproductive performance.

Activin A and follistatin during the oestrous cycle and early pregnancy in ewes.

The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES.

Mutations in the leptin receptor gene associated with delayed onset of puberty are also associated with decreased ovulation and lambing rates in prolific Davisdale sheep.

The aim of this study was to determine if single nucleotide polymorphisms (SNPs) in the leptin receptor (LEPR) gene associated with delayed onset of puberty are associated with changes in other reproductive traits in adult ewes. The ovulation rate of ewes homozygous for the SNPs was ~15% lower (PPLEPR SNPs than their wild-type or heterozygous contemporaries. Partial failure of multiple ovulations was also increased (PLEPR had on average 0.2 fewer lambs at mid-pregnancy and at birth compared with the wild-type or heterozygous ewes (PLEPR were strongly associated with poorer reproductive performance in Davisdale ewes, which is likely to be linked to both a reduced number of ova available for fertilisation and an increased number of ewes failing to become pregnant. Increased partial failure of multiple ovulations in ewes with high ovulation rates (i.e. 3 or greater) may also contribute to the poor reproductive performance.

Single-nucleotide polymorphisms in the LEPR gene are associated with divergent phenotypes for age at onset of puberty in Davisdale ewes.

Attainment of puberty is a key developmental event influenced by genetic and environmental factors. In examining age at attainment of puberty, we observed closely related rams from the Davisdale line whose daughters differed in age at which they attained puberty. A candidate gene approach was used to identify mutations that may underlie these observed differences. Four rams with divergent phenotypes for their daughter's age at onset of puberty were selected for whole-genome sequencing. The coding regions of genes with known roles in regulating reproductive function were searched for single-nucleotide polymorphisms (SNPs) that altered the amino acid sequence of the protein. Of interest were three SNPs in the leptin receptor gene (LEPR). A Sequenom assay was developed to determine the genotype of these SNPs in daughters of 17 sons of a founding sire. A higher percentage of ewe lambs homozygous for the LEPR mutations failed to undergo puberty before 1 yr of age, and those that did undergo puberty during the first breeding season on average were approximately 17 days older than homozygous wild-type ewes. Heterozygous ewes were intermediate for both measurements. Given the predicted change in protein function produced by the mutation in LEPR and the strong associations between the genotype and onset of puberty phenotypes, we propose that this mutation in LEPR underlies the observed difference in age at onset of puberty in the Davisdale line. Furthermore, these animals will likely provide a useful model to better understand the role of leptin in the regulation of puberty.

Effects of immunizing ewes against bone morphogenetic protein 15 on their responses to exogenous gonadotrophins to induce multiple ovulations.

Sheep with a heterozygous inactivating mutation in the bone morphogenetic protein 15 (BMP15) gene experience an increased ovulation rate during either a natural oestrous cycle or a cycle in which exogenous FSH and eCG (gonadotrophins) are given to induce multiple ovulations. The primary aim of these studies was to determine whether ewes immunised against BMP15 would also show an improved superovulation rate following exogenous gonadotrophin treatment. A secondary aim was to determine the effects of BMP15 immunisation on ovarian follicular characteristics. In most ewes (i.e. > 75%) immunised with a BMP15-keyhole limpet haemocyanin peptide in an oil-based adjuvant in order to completely neutralise BMP15 bioactivity, there was no superovulation response to exogenous gonadotrophins. In ewes treated with exogenous gonadotrophins following a BMP15-BSA peptide immunisation in a water-based adjuvant to partially neutralise BMP15 bioactivity, the ovulation rate response was similar to the control superovulation treatment groups. Characterisation of follicular function revealed that the water-based BMP15-immunised animals had fewer non-atretic follicles 2.5-3.5 or > 4.5  mm in diameter compared with controls. Basal concentrations of cAMP were higher in granulosa cells from animals immunised against BMP15 than control animals. There were no significant differences in the concentrations of cAMP between granulosa cells from BMP15- and control-immunised animals when given FSH or hCG, although there were differences in the proportions of follicles in different size classes that responded to FSH or hCG. Thus, immunisation against BMP15 may have been causing premature luteinisation and thereby limiting the numbers of follicles recruited for ovulation following treatment with exogenous gonadotrophins.

Identification of a line of sheep carrying a putative autosomal gene increasing ovulation rate in sheep that does not appear to interact with mutations in the transforming growth factor beta superfamily.

Sheep lines with mutations in single genes that have major effects on ovulation rate have been very useful in gaining a better understanding of pathways important in controlling follicular development and ovulation rate. To date however, all known mutations are in the transforming growth factor beta (TGFB) superfamily. Ovulation rates were measured in 720 progeny of 20 rams that were descendants of a single prolific ewe. Evaluation of ovulation rates of daughters of closely related sires suggests the presence of a segregating major gene Fecundity Davisdale (FECD) that increases ovulation rate between 0.4 and 0.8 in heterozygous daughters. Key features of mutations in genes of the TGFB superfamily pathway, such as synergistic interactions with other family members, infertility in homozygous carriers, and increased responsiveness to exogenous gonadotropins, were not observed in this line; thus, the mutation does not appear to be acting in the TGFB pathway. Hence, there is likely a novel mutation being carried in this line of sheep that alters ovulation rate. Future identification of the causative mutation may provide new insights into regulation of follicular development and ovulation rate.