Increased Ovulation Rate Research Articles

Plane of nutrition during the rearing phase for replacement ewes of four genotypes: I – effects on growth and development, and on ovulation rate at first joining

Plane of nutrition (PN) offered to ewe replacements during the rearing phase (8 to 17 months) affects BW at joining and may affect reproductive performance when joined at ~19 months. The effects of PN offered to ewe replacements during their first winter (winter_1) and second summer (summer_2) were evaluated over 3 consecutive years, using 287 spring-born ewe lambs, representing four genotypes (Belclare (Bel), Charmoise×Scottish Blackface (C×SBF), Bel×SBF (Bel×SBF), Bel×SBF that were heterozygous either FecGH or FecXG mutations that increase ovulation rate (OR) (BelMG×SBF)). During extended (deferred) grazing in winter_1 (November to March) the lambs were offered herbage DM allowances (HA) of 0.75 (L) or 1.75 (H) kg/day. During summer_2 (March to August) the replacements were set stocked to maintain sward heights of 4 (L) or 6 (H) cm, thus yielding a 2×2 factorial design for the nutrition treatments (71 to 73 animals/treatment). Increasing HA during winter_1, and residual sward height during summer_2, increased (P<0.001) BW and body size at joining (19 months) but had no effect (P>0.05) on OR. Mean BW at joining was 53.3, 56.8, 56.6 and 61.7 (SEM 0.74) kg for ewes offered the LL, LH, HL and HH treatments, respectively. Mean OR of C×SBF and BelMG×SBF differed (P<0.001) from that of Bel×SBF by −0.58 and +0.47, respectively. Correlations between linear measurements (LMs) of body size and BW at 7, 12 and 17 months were all positive and significant (P<0.001). The precision of the set of LMs as a predictor of BW at joining improved with age (R2 0.46, 0.54 and 0.74 at 7, 12 and 17 months) but BW at a given age was a better predictor at all age time points. Chest girth was the best predictor, among the LMs, of BW at joining and its explanatory power increased with age. Equations developed using LMs at 7, 12 or 17 months, to predict BW at joining exhibited biases of −2.1, −1.5 and +0.9 kg, respectively. It is concluded that whilst altering PN during the rearing phase changed BW by 16% it had no effect on OR. There was no interaction (P>0.05) between the PN offered during winter_1 and summer_2, or PN and ewe genotype for BW at joining or OR. LMs, either at 7, 12 or 17 months of age, are useful predictors of BW at joining.

Control of oestrous cycle in small ruminants

Control of oestrous cycle is an important management tool in sheep and goat farms. Over the years, various methods have been developed and many applications have been documented in the international literature. In this review paper, the theoretical background and practical aspects of oestrous cycle control in small ruminants are reviewed; furthermore, problems related to oestrous cycle control are discussed. The article is divided in the following sections: (i) Methods for the control of the oestrous cycle (selection of method, male effect, pharmaceutical methods), (ii) Methods for increasing ovulation rate (nutrition, genetics, hormonal treatment, immunization techniques), (iii) Induction of early puberty, (iv) Fertilization of animals (mating or artificial insemination), (v) Problems during application of oestrous control methods (side effects of treatments, application errors).

Trio, a novel high fecundity allele: I. Transcriptome analysis of granulosa cells from carriers and noncarriers of a major gene for bovine ovulation rate.

A major gene for bovine ovulation rate has been mapped to a 1.2 Mb region of chromosome 10. Screening of coding regions of positional candidate genes within this region failed to reveal a causative polymorphism, leading to the hypothesis that the phenotype results from differences in candidate gene expression rather than alteration of gene structure. This study tested differences in expression of positional candidate genes in granulosa cells between carriers and noncarriers of the high fecundity allele, as well as characterizing differences in the transcriptomic profile between genotypes. Five carriers and five noncarriers, female descendants of "Trio," a carrier of the high fecundity allele were initially used in an RNA-seq analysis of gene expression. Four of ten samples were contaminated with theca cells, so that six samples were used in the final analysis (three of each genotype). Of 14 973 genes expressed, 143 were differentially expressed (false discovery rate P < 0.05) in carriers versus noncarriers. Among the positional candidate genes, SMAD6 was 6.6-fold overexpressed in the carriers compared to noncarriers (P < 5 × 10-5). This result was replicated in an independent group of 12 females (7 carriers and 5 noncarriers) using quantitative real-time PCR; SMAD6 was 9.3-fold overexpressed in carriers versus noncarriers (P = 1.17 × 10-6). Association of overexpression of SMAD6, an inhibitor of the BMP/SMAD signaling pathway, with high ovulation rate corresponds well with disabling mutations in ligands (BMP15 and GDF9) and a receptor (BMPR1B) of this pathway that cause increased ovulation rate in sheep.

The key role of targeted betacarotene supplementation on endocrine and reproductive outcomes in goats: Follicular development, ovulation rate and the GH-IGF-1 axis

The possible effects of betacarotene (BC) supplementation on the secretion pattern of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), and their possible relationship with total ovarian activity (TOA), was evaluated in adult goats during the breeding season. In October, goats [n=22, 3.5y. old, 7/8 Sannen-Alpine] were randomly assigned to: a) Betacarotene group [BC, n=10; 45.9±1.97kg live weight (LW), 3.04±0.08 units, body condition score (BCS), supplemented with 50mg of BC goat day−1], and b) Control group [CONT, n=12; 46.2±2.04kg LW, 3.0±0.08 units, BCS]. An ultrasonographic scan was performed to evaluate corpus luteum number (OR) and antral follicle number (AF); TOA=OR+AF. Average LW and BCS did not differ (p>0.05) during the experimental period, yet BC-goats reflected an increased OR (3.4±0.2 vs. 2.8±0.2), AF (5.0±0.6 vs. 3.4±0.6) and TOA (8.4±0.5 vs. 6.2±0.6). Regarding the endocrine profile, the lowest (p<0.05) serum GH average concentrations (10.0 vs. 14.3±1.0ngmL−1; p=0.01) and GH-AUC (3670.4 vs. 5235.7±369.8 units; p=0.01), were observed in the BC-supplemented group. Neither serum IGF-1 concentrations (254.6±28.9ngmL−1 p>0.05) nor GH-PULSE (1.4±0.5 pulses 6h−1 p>0.05) differed between treatments. We document a potential role of BC as modulator of somatotrophic function, decreasing mean serum concentration and the area under the curve of GH, while also noting a positive action upon ovarian function with increases in ovulation rate and antral follicular development; such outcomes may embrace not only physiologic significance but also potential translational applications.

Effect of GnRH on ovulatory response after luteolysis induced by two low doses of PGF2α in lactating dairy cows

This study investigated the effect of gonadotropin-releasing hormone (GnRH) on ovulatory response after complete luteolysis induced by two low doses of prostaglandin (PG)F2α in lactating dairy cows. Cows (n = 18) ranging between 45 and 65 days in milk were recruited for synchronization by a modified Ovsynch-48 protocol (GnRH-7 days-375 μg PGF2α-1 day-250 μg of PGF2α-1 day-GnRH) over a total of 23 estrous cycles. Synchronized cows (n = 16) were randomly assigned to GnRH and Saline groups in stage 1 of the experiment after 9–10 days of ovulation in synchronization. On days 0 and 1 (day 0 = first PGF2α administration), cows were treated with 375 and 250 μg PGF2α, respectively. On day 2, cows in the GnRH and Saline groups were administered 250 μg GnRH or 2.5 mL of 0.9% saline, respectively. Serum progesterone (P4) levels were measured and changes in the corpus luteum (CL) were ultrasonically monitored daily from day 0–3 to assess complete luteolysis. Preovulatory follicle diameter and ovulatory response were evaluated by ultrasonography. In stage 2, cows were treated in a manner converse to that in stage 1. The synchronization rate was 69.6% (16/23). In stages 1 and 2, cows showed complete luteolysis with P4 concentration <1 ng/mL or remaining CL area <50%. Average ovulation time was 29.3 ± 0.5 h, which mostly occurred between 28 and 30 h after GnRH injection. However, all cows in the Saline group ovulated later than 36 h post-injection, with an average time of 52.7 ± 8.6 h. There was no difference in preovulatory follicle diameter between the two groups (16.8 ± 0.5 and 17.3 ± 0.5 mm for GnRH and saline groups, respectively). Although ovulation rate was not correlated with treatment, the rate within 48 h of GnRH injection (93.3%) tended to be higher compared with that in the Saline group (60.0%). Thus, GnRH administration increased ovulation rate following complete luteolysis induced by two low doses of PGF2α. These results indicate that this simple protocol for dairy cows is an effective alternative to timed artificial insemination programs in the field.

Ovulation rate, antral follicle count, and circulating anti-Müllerian hormone in Trio allele carriers, a novel high fecundity bovine genotype

High fecundity genotypes in sheep are a valuable model to study the physiological mechanisms underlying follicle selection and the control of ovulation rate. Similar genotypes in cattle had not been described until the recent identification of a major bovine allele, termed Trio, which had a large effect on ovulation rate. The present study was designed to evaluate ovulation rate, antral follicle count (AFC), circulating ant-müllerian hormone (AMH), and the association among these measures in unstimulated and superstimulated Trio carrier cattle. We hypothesized that AFC and AMH would be variable among individual cows but would be similar between Trio carriers and non-carrier control cows and that there would be no association between these measures of follicle numbers and ovulation rate. In experiment 1, ovulation rate was determined during 4 consecutive estrous cycles in Trio carriers (n = 34) and non-carrier controls (n = 27). Ovulation rate, on average, was greater (P < 0.01) in Trio carriers (3.5 ± 0.2) compared to non-carrier controls (1.1 ± 0.1) with ∼70% of carrier cycles (n = 136) having 3–4 ovulations while only ∼5% had single ovulations. In contrast, non-carrier cycles (n = 108) were mostly single ovulation (89%) with none having more than two ovulations. In experiment 2, AFC, determined at wave emergence, was not different (P = 0.54) between Trio carriers (24.5 ± 1.3; n = 45) and non-carrier controls (23.1 ± 0.9; n = 37), and no correlation was found between AFC and mean ovulation rate in either genotype (r = −0.009 and r = −0.07; P > 0.70, respectively). In Experiment 3, circulating AMH was also not different between genotypes (P = 0.65) while correlations were found between AFC and AMH in Trio carriers (r = 0.43; P = 0.05; n = 27) and non-carrier controls (r = 0.78; P < 0.01; n = 19). In experiment 4, AFC and AMH were determined in Trio-carriers (n = 9) in relation to a synchronized follicular wave which was unstimulated or stimulated with exogenous FSH. Stimulation with FSH increased ovulation rate, compared to unstimulated Trio carriers, however no association was found between AFC or AMH and ovulation rate regardless of whether superstimulation with exogenous FSH was used. In conclusion, the novel high fecundity bovine genotype Trio, results in consistent multiple ovulations despite having similar AFC and AMH. Therefore, our results suggest that differences in antral follicle numbers during the final stages of follicle development are not a key component of the mechanism underlying multiple ovulations in Trio carriers.

FecXBar a Novel BMP15 mutation responsible for prolificacy and female sterility in Tunisian Barbarine Sheep

BackgroundNaturally occurring mutations in growth and differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) genes are associated with increased ovulation rate (OR) and litter size (LS) but also sterility. Observing the Tunisian Barbarine ewes of the “W” flock selected for improved prolificacy, we found prolific and infertile ewes with streaky ovaries. Blood genomic DNA was extracted from a subset of low-ovulating, prolific and infertile ewes of the “W” flock, and the entire coding sequences of GDF9 and BMP15 were sequenced.ResultsWe evidenced a novel polymorphism in the exon 1 of the BMP15 gene associated with increased prolificacy and sterility. This novel mutation called FecXBar is a composite polymorphism associating a single nucleotide substitution (c.301G > T), a 3 bp deletion (c.302_304delCTA) and a C insertion (c.310insC) in the ovine BMP15 cDNA leading to a frame shift at protein position 101. Calculated in the “W” flock, the FecXBar allele increased OR by 0.7 ova and LS by 0.3 lambs (p = 0.08). As for already identified mutations, homozygous females carrying FecXBar exhibited streaky ovaries with a blockade at the primary stage of folliculogenesis as shown by histochemistry.ConclusionsOur investigation demonstrates a new mutation in the BMP15 gene providing a valuable genetic tool to control fecundity in Tunisian Barbarine, usable for diffusion program into conventional flocks looking for prolificacy improvement.

Effect of corifollitropin alfa supplemented with or without LH on ovarian stimulation and embryo viability in rabbit

There is increasing interest in using rabbits for research as a laboratory model as well as for industrial production of meat, wool and fur. Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to either support genetic improvement programs, ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, posology and the effect of gonadotropins used simultaneously, the donor and the environment. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (CTP), alone or supplemented with LH, versus a FSH standard protocol of five equal doses administered twice daily to superovulate rabbit does (20 per group and 29 control females). We determined: 1) the impact of this stimulation on in vitro development and mRNA expression at blastocyst stage and 2) in vivo embryo development and viability rate at birth of transferred embryos. Our outcomes showed that the ovulation rate was similar among the different ovarian stimulation groups, reaching more than fourfold the ovulation rate of a control doe. While rates of embryos developing to the blastocyst stage after 48 h of in vitro culture were similar between groups, the hatched blastocyst rate was higher for superovulated embryos from CTP group. Moreover, no significant differences among mRNA expression of OCT4, SOX2 and NANOG genes were detected. Nevertheless, embryos from ovarian stimulated does with CTP + LH showed significantly higher implantation rates and survival at birth among the different ovarian stimulation groups and similar to those in the control group. In conclusion, the results of this study suggest that a single injection of long acting corifollitropin alfa can be effectively used in rabbits to elicit a more than fourfold increase in ovulation rate compared to control animals. In addition, the LH supplementation allows us to obtain similar in vivo embryo development results as in the control group.

BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep.

Polymorphisms in the gene encoding bone morphogenetic protein 15 (BMP15) have been associated with multiple ovulations in sheep. As BMP15 regulates inhibin expression in rodents, we assumed that the ovarian inhibin/activin system could mediate part of the effect of BMP15 mutations in the regulation of ovulation rate in sheep. To answer this question, we have studied the effects of two natural loss-of-function mutations of BMP15 on the expression of components of this system. The FecXR and the FecXGr mutations, when present respectively in Rasa Aragonesa ewes at the heterozygous state and in Grivette ewes at the homozygous state, were associated with a twofold increase in ovulation rate. There were only small differences between mutant and wild-type ewes for mRNA expression of INHA, INHBA, ACVR1B, ACVR2A, FST or TGFBR3 in granulosa cells and inhibin A or activin A concentrations in follicular fluid. Moreover, the effects of mutations differed between breeds. In cultures of granulosa cells from wild-type ewes, BMP15, acting alone or in synergy with GDF9, stimulated INHA, INHBA and FST expression, but inhibited the expression of TGFBR3 Activin A did not affect INHBA expression, but inhibited the expression of ACVR2A also. The complexity of the inhibin/activin system, including positive and antagonistic elements, and the differential regulation of these elements by BMP15 and activin can explain that the effects of BMP15 mutations differ when present in different genetic backgrounds. In conclusion, the ovarian inhibin/activin system is unlikely to participate in the increase of ovulation rate associated with BMP15 mutations in sheep.

The "regulating conception-governor vessel" acupuncture method for infertility of polycystic ovarian syndrome

To compare clinical efficacy differences between"regulating conception-governor vessel" acupuncture method and clomiphene for infertility of polycystic ovarian syndrome (PCOS). One hundred patients of PCOS were randomly assigned into an observation group and a control group, 50 cases in each one. The patients in the observation group were treated with"regulating Conception-Governor Vessel" acupuncture method at Zhongwan (CV 12), Guanyuan (CV 4), Qihai (CV 6), Zhongji (CV 3), Mingmen (GV 4), Yaoyangguan (GV 3) and Yaoshu (GV 2). The acupuncture treatment started at the end of menstruation, once every other day; after four treatments, the follicle was tested with B ultrasound; when follicle was longer than 18 mm or above, the acupuncture treatment was given once a day until follicle was discharged and acupuncture treatment finished. The patients in the control group were treated with oral administration of clomiphene from 5 days into menstruation, 50 mg per day for 5 consecutive days. Both groups were treated for three menstruation cycle. The menstrual cycles, endometrial thickness, endometrium types (A, B and C), cervical mucus scores, basal body temperature (BBT), cases of ovulation, cases of pregnancy were observed.The levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), prolactin (PRL), progesterone (P) and testosterone (T) in serum were detected before and after the treatment. After the treatment, the menstrual cycles were shortened (both P<0.05),the endometrial thickness and cervical mucus scores were increased in the two groups (P<0.05, P<0.01); the improvement of endometrial thickness and cervical mucus score in the observation group were higher than those in the control group (both P<0.01). The percentages of type-A endometrium in both groups were higher than those before the treatment (both P<0.05); the number of type-A endometrium in the observation group was higher than that in the control group (P<0.05). The ovulation rate was 88.0% (44/50) in the observation group, which was superior to 70.0% (35/50) in the control group (P<0.05). After treatment, the levels of LH were reduced in the two groups (P<0.05, P<0.01), which was more significant in the observation group (P<0.05). The"regulating Conception-Governor Vessel" acupuncture method could improve menstrual cycles, increase endomet-rial thickness and promote the development of follicles in PCOS patient; in addition, it could decrease serum LH level, improve the ovarian functions and increase ovulation rate, which is superior to oral administration of clomiphene.

Disruption of the sheep BMPR-IB gene by CRISPR/Cas9 in in vitro-produced embryos

BMPR-IB (also known as FecB) is a key candidate gene for the genetic control of sheep reproductive performance. Loss-of-function mutations in the sheep BMPR-IB gene lead to an increase in ovulation rate and consequently larger litter size. However, the BMPR-IB gene has been identified in only a few sheep breeds. To improve sheep reproduction through modification of the BMPR-IB gene, we designed an sgRNA to target the sheep BMPR-IB gene by using the CRISPR/Cas9 system. First, we performed gene editing by injecting Cas9/sgRNA into the cytoplasm of one-cell fertilized eggs. A total of 88 embryos were assayed by T7EI digestion and Sanger sequencing. The results reported that the efficiency of gene modification was 37.5% (33/88) and increased with the developmental stage of embryo from the 2-cell stage to the blastocyst stage. Of the 33 gene editing embryos, 12 (36%, 12/33) were homozygous and 21 (64%, 21/33) were heterozygous. Moreover, sequence analysis of the PCR products from the positive embryos revealed that there were more than 10 modification forms that resulted in frame shift and truncated proteins. Further analysis by cloning and sequencing of each individual embryo showed a high level of mosaicism. In addition, off-target event analysis revealed that none of the off-target mutations was introduced into the embryos. Our results indicated that the Cas9/sgRNA system is a simple and efficient tool that may potentially be used in the genetic modification of the sheep BMPR-IB gene in vivo.

Short-term supplementation with maize increases ovulation rate in goats when dietary metabolizable energy provides requirements for both maintenance and 1.5 times maintenance

This study aimed to evaluate ovarian follicular dynamics in goats submitted to synchronization of estrus and supplemented with diets that differed in the metabolizable energy source and amount of energy. The experiment was carried out using 42 does allocated into three treatments, fed for 9 days with a ration providing 1.0 times maintenance containing maize (1 MM, n = 14) or without maize (1 M, n = 14) or a ration providing 1.5 times maintenance containing maize (1.5 MM, n = 14). Estrus was synchronized with two injections of cloprostenol given 7 days apart. Does were also treated with intravaginal progesterone inserts and eCG. The number of ovulations and size of the follicles were measured using ultrasonography on Days 10, 11, and 12 after the start of the dietary treatment. The interval to estrus and duration of estrus did not differ between treatments (P = 0.382). Does fed with 1 and 1.5 MM had a similar number of ovulations but a greater number of ovulations than goats fed with 1 M (P = 0.028). The mean number of small, medium, large, and total number of follicles on Days 10 to 12 of ultrasound evaluations did not differ (P = 0.204) between treatments, but mean numbers changed over time (P < 0.001). The mean frequency and amplitude of LH pulses and concentrations of glucose, insulin, leptin, and insulin growth factor-1 in plasma were not significantly affected (P > 0.258) by any of the treatments. In summary, the inclusion of maize in the ration can stimulate ovulation rate at maintenance level. Similar results between groups fed diets that included maize and provided metabolizable energy at 1.0 and 1.5 maintenance demonstrate that to increase the ovulation rate when synchronizing estrous cycles in does, dietary supplementation with maize can be restricted to provide a maintenance level of metabolizable energy only, which would reduce dietary costs.

Estudios realizados y situación actual de la variante génica prolífica ROA (FecXR) de la raza ovina rasa aragonesa

In the present work we present the main productive studies carried out to characterize ROA sheep (R+, Rasa Oviaragon®), ewes of the Rasa Aragonesa breed carrying in heterozygosity a natural genetic prolific variant found in 2007 (FecXR allele, BMP15 gene). Its effect on prolificacy leads to an increase of 0.35 lambs/lambing ewe when compared with non-carrier ewes, with an additive effect over the standard hormonal treatments used in farms. In artificially inseminated ewes, an increased fertility was also found in a set of farms (71.1 vs. 59.5 %; p<0.01) as well as an advanced puberty (around 30 days). On the contrary, no differences between genotypes were found in male’s fertility. The studies on the preovulatory LH peak did not show differences between genotypes, which allows for the application of the same protocols used in non-carrier ewes concerning Artificial Insemination (AI) at fixed time. The increased ovulation rate of R+ ewes could be related with the smaller size of their ovarian follicles, with no further influence on fertility, as sown in farms as well as on in vitro embryo production studies. Regarding lamb marketing, the presence of the FecXR allele does not affect morphology, lamb growth or the characteristics of the carcass or meat quality of their offspring. Finally, it has been observed that the productive advantages of the FecXR (ROA) variant result in an increase in profitability of farms that use this variant, which confirms its interest for most farmers.

Effect of increased ovulation rate on embryo and foetal survival as a model for selection by ovulation rate in rabbits

Selection for ovulation rate in prolific species has not improved litter size, due to an increase in prenatal mortality, with most mortality observed in the foetal period. The aim of this study was to investigate the magnitude and timing of embryo and early foetal survival in females with high ovulation rate using hormonal treatment as a model for selection by ovulation rate. Two groups of females (treated and untreated) were used. Treated females were injected with 50 IU equine chorionic gonadotropin 48 h before mating. Females were slaughtered at 18 d of gestation. Ovulation rate (OR), number of implanted embryos (IE), number of live foetuses at 12 and 18 d (LF&lt;sub&gt;12&lt;/sub&gt; and LF&lt;sub&gt;18&lt;/sub&gt;, respectively) were recorded. In addition, embryo survival (ES=IE/OR), foetal survival at 18 d of gestation (FS&lt;sub&gt;LF18&lt;/sub&gt;=LF&lt;sub&gt;18&lt;/sub&gt;/IE), foetal survival between 12 and 18 d of gestation (FS&lt;sub&gt;LF18/LF12&lt;/sub&gt;=LF&lt;sub&gt;18&lt;/sub&gt;/LF&lt;sub&gt;12&lt;/sub&gt;) and prenatal survival (PS&lt;sub&gt;LF18&lt;/sub&gt;=LF&lt;sub&gt;18&lt;/sub&gt;/OR) were estimated. For each female, the mean and variability of the weight for live foetuses (LFWm and LFWv, respectively) and their placentas (LFPWm and LFPWv, respectively) were calculated. Treated females had a higher ovulation rate (+3.02 ova) than untreated females, with a probability of 0.99. An increase in the differences (D) between treated and untreated females was observed from implantation to 18 d of gestation (D=–0.33, –0.70 and –1.28 for IE, LF&lt;sub&gt;12&lt;/sub&gt; and LF&lt;sub&gt;18&lt;/sub&gt;, respectively). These differences had a low accuracy and the probability that treated females would have a lower number of foetuses also increased throughout gestation (0.60, 0.70 and 0.86 for IE, LF&lt;sub&gt;12&lt;/sub&gt; and LF&lt;sub&gt;18&lt;/sub&gt;, respectively). According to the previous results for OR and LF&lt;sub&gt;18&lt;/sub&gt;, treated females showed a lower survival rate from ovulation to 18 d of gestation (D=–0.12, P=0.98 for PS&lt;sub&gt;LF18&lt;/sub&gt;). Treated females also had lower embryo and foetal survival (D=–0.10 and P=0.94 for ES and D=–0.08 and P=0.93 for FS&lt;sub&gt;LF18&lt;/sub&gt;). Main differences in foetal survival appeared from 12 to 18 d of gestation (D=–0.09 and P=0.98 for FS&lt;sub&gt;LF18/LF12&lt;/sub&gt;). Unexpectedly, treated females showed similar foetus weight and higher foetal placenta weight than untreated females (D=0.25 g, P=0.98) and lower variability for these traits (D=–0.02 g, P=0.72 for LFWv and D=–0.05 g, P=0.83 for LFPWv). These results are not related to a lower number of IE or LF&lt;sub&gt;18&lt;/sub&gt;. Thus, the effect of increasing by three ova in rabbits leads to a lower embryo and early foetal survival. There seems to be no relationship between foetal mortality and foetus weight.

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

Effect ofFecBGene on Body Weight in Black Bengal Goat

FecB gene is first described gene which has been found to increase ovulation rate and litter size in sheep. But, work related to genetic mechanism and genetic markers for caprine proliferation has not been done so much. The present study was aimed to screen Black Bengal goat population for polymorphism of FecB gene and to study its effect on body weight at different growth stages. DNA samples from 96 animals were isolated and subjected to PCR. Amplified fragments obtained were allowed for polyacrylamide gel electrophoresis for the detection of single strand conformational polymorphism (SSCP) variants. Among all samples, three different SSCP variants were found which were marked as AA, AB and BB. The highest genotype frequency was observed for AB (0.38), which was followed by BB (0.33) and AA (0.29). Least-square analysis of variance showed significant (P<0.01) effect of genotype on body weight at birth only. The least square mean of body weights at birth for genotype BB (01.17±00.03 kg) was significantly lower than that of genotype AA (01.43±00.03 kg) and genotype AB (01.36±00.02 kg). It was also observed that genotype had non-significant effect on body weight at 4-week, 8-week, 12-week, 24-week, 36-week and 48-week of age. These results indicate that effect of FecB on body weight was limited at birth only. No significant effects were found on growth rate at later stage of life. FecB gene was hitherto linked to prolificacy. So, the effect of FecB gene on other traits has important bearing if the research is continued further with more number of species specific primers at other loci.

Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep

The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations.

The effects of intravenous, glucose versus saline on ovarian follicles and their levels of some mediators of insulin signalling.

BackgroundA short-term increase in food intake and specifically dietary energy can stimulate folliculogenesis and increase ovulation rate in ewes. The mechanism appears to involve the insulin-glucose metabolic system and its interaction with FSH signalling pathways in the granulosa cells of ovarian follicles. This experiment was designed to investigate the interaction between these two systems in the granulosa cells of ovarian follicles.MethodsThirty six Ile-de-France ewes were used in this controlled experiment to study the effects of intravenous glucose on folliculogenesis. Eighteen ewes were infused with glucose (10 mM/h for 72 h) from day 8 of the oestrous cycle, while the others (controls) received saline. Ovaries were collected when the infusions ended (luteal phase) or 30 h later and after a luteolytic dose of a PGF2α analogue (follicular phase). Follicles were dissected and granulosa cells and follicular fluid harvested. The blood concentrations of glucose, insulin, oestradiol and FSH were monitored over the experiment. The levels of Aromatase P450 and of the phosphorylated and non-phosphorylated forms of Akt, AMPK and ERK in granulosa cells and the concentration of oestradiol in follicular fluid, were determined.ResultsGlucose increased the circulating concentration of glucose (P < 0.05) and insulin (P < 0.05). It also increased the total number of follicles >1.0 mm in diameter (P < 0.05) and small (P < 0.05) follicles (>1.0 to 2.0 mm in diameter) but not medium (>2.0 to 3.5 mm in diameter) or large (>3.5 mm in diameter) follicles. Glucose decreased circulating oestradiol (P < 0.05) but not that of FSH or progesterone. Glucose reduced aromatase P450 (P < 0.05) and decreased the phosphorylation of Akt (P < 0.05), ERK (P < 0.05) and AMPK (P < 0.05) in granulosa cells from oestrogenic follicles. The level of Aromatase P450 was greatest in large oestrogenic follicles and the phosphorylation of Akt (P < 0.05), ERK (P < 0.05) and AMPK (P < 0.05) was lower in small follicles compared to medium and large follicles.ConclusionsThese data suggest that the effect of glucose in small follicles is a direct action of glucose that increases the number of small follicles while the effect of glucose in oestrogenic follicles is an indirect insulin-mediated action.

177 DOWN-REGULATION OF P450 AROMATASE AND GLUCOSE TRANSPORTER 4 mRNAs EXPRESSION IN SHEEP OVARIAN FOLLICLES AFTER ULTRASHORT NUTRITIONAL FLUSHING

Nutritional supplementation before breeding (flushing) has become a common practice and is a reliable method to increase lambing and twining rates in sheep. We have shown that ultrashort flushing (USF: 1 day long) acutely increases ovulation rate without affecting the body condition or animal weight. A short nutritional stimulus would necessarily be acting either directly at the ovarian level or through hormones and factors other than gonadotropins. The possible mechanisms of action of USF on ovulation rate is the deregulation of the feedback loop between the ovaries and gonadotropin secretion, where nutrition causes a direct inhibition of follicular oestradiol production leading to compensatory secretion of FSH that stimulates folliculogenesis, or direct stimulation of follicular development through hormones and factors other than gonadotropins. We hypothesised that USF enhances the number of follicles selected for ovulation due to either a decrease in the expression of P450 aromatase or alternatively by advancing the maturation of the ovarian follicles, which would be reflected in an increase in LH receptor (LHr) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression, an increase in glucose transporter 4 (GLUT4) mRNA expression, or both. To test these hypotheses, the oestrus cycle of 30 ewes was synchronized with progestin intravaginal sponges and prostaglandins. Animals were given the USF on the day of progestin withdrawal. The ovaries were removed surgically before treatment (time 0; n = 6), or at 12, 24, and 48 h after being treated with either glycerol or water (n = 4 per treatment by time category). The ovarian follicles were dissected out and counted. Follicles larger than 3 mm in diameter were pooled and the mRNA extracted for specific P450aromatase, 3β-HSD, LHr, and glucose transporter 4 determinations. USF increased the number of follicles larger than 3 mm at 48 h after treatment (P &lt; 0.05). No effect was observed in the number of small follicles. In the two largest follicles, aromatase and GLUT4 mRNA abundance decreased (P &lt; 0.01) 12 h after treatment. There was no effect of treatment on mRNA for LH receptor or 3β-HSD. These results demonstrate a reduction in aromatase expression in potentially ovulatory follicles 12 h after flushing. The decrease in aromatase may favour a transient increase in FSH concentrations 12 h after flushing that would allow the stimulation and selection of supplementary ovulatory follicles.

Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes.

Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P<0.001), averaging 2.1±0.1 when compared to 1.2±0.1 in wild-type ewes. The OR was also influenced by age, increasing in the second and third breeding seasons (P<0.001). In flocks segregating this allele, the LS was higher in mutant sheep (P<0.001), averaging 1.61±0.07 in heterozygotes and 1.29±0.03 in wild-type ewes. Analysis of homozygote reproductive tract morphology revealed uterine and ovarian hypoplasia. Ovarian follicles continue to develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post-translation processing and the fate of the follicular cells that remain after the oocyte demise.