Antonio Gonzalez-Bulnes

Multiple factors affecting the efficiency of multiple ovulation and embryo transfer in sheep and goats

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

Measurement of inhibin A and follicular status predict the response of ewes to superovulatory FSH treatments.

Variability in superovulatory response to FSH stimulation is common to most mammals and imposes practical problems for assisted reproduction. In sheep, we have studied if this response is related to the ovarian follicular population and activity before the stimulation. During the breeding season, 30 ewes were treated with 40 mg FGA sponges for 14 days and 125 microg cloprostenol injection on Day 12, considering Day 0 as the day of progestagen insertion. Superovulatory response was induced with two different FSH regimes using the same total dose (8.8 mg), administered twice daily from 60 h before to 24 h after progestagen withdrawal. At the first FSH injection, all follicles > or = 2 mm were observed by transrectal ultrasonography and plasma FSH and inhibin A levels were determined. The number of corpora lutea and the number of and viability of recovered embryos in response to the treatment were determined on Day 7 after sponge withdrawal. No significant differences were found between treatments. The total mean number of corpora lutea (11.5 +/- 1.2) and recovered embryos (7.9 +/- 1.1) were positively correlated (P < 0.05 and <0.01, respectively) with the number of small antral follicles (2-3 mm: 9.2 +/- 0.7) and inhibin A concentration (240 +/- 18 pg/ml; P < 0.05 for corpora lutea and P < 0.005 for recovered embryos) observed at the onset of the superovulatory treatment, which was also positively correlated with the number of viable embryos (5.8 +/- 0.9, P < 0.005). In 18 ewes with follicles > or = 6 mm prior to FSH treatment, the ovulation rate was unaffected but the number of embryos (6.1 +/- 0.9 versus 11.6 +/- 2; P < 0.05) and their viability (4.5 +/- 0.8 versus 8.5 +/- 2; P < 0.05) was reduced. The lower number of embryos produced when a large follicle is present suggest that a proportion of the smaller follicles are in early stages of atresia and the developmental competence of their oocyte is compromised.

Origin of the preovulatory follicle in Mouflon sheep (Ovis gmelini musimon) and effect on growth of remaining follicles during the follicular phase of oestrous cycle

Daily transrectal ultrasonographies were conducted to study development of all follicles with antral diameters > or = 2mm during the follicular phase of oestrous cycle in Mouflon, a strictly monovular wild-sheep. A total of 14 follicular phases was studied after oestrus synchronization with two cloprostenol doses, 9 days apart, in five cyclic Mouflon ewes. In 13 cycles (92.8%), the ovulatory follicle arose from those antral follicles present in both ovaries when luteolysis was induced, being the largest one with a mean size of 4.4+/- 0.3mm at that moment in 10 cycles (76.9%). The remaining cycles had a larger follicle, but it was decreasing in size. Appearance of new follicles > or =2mm in size remained unaffected during the follicular phase (3.7+/- 0.2), but there was found a linear decrease in the number of those growing to > or =3mm (2.5+/- 0.4 to 1.1+/- 0.2, P < 0.05) and > or = 4mm (0.6+/- 0.2 to 0.1+/- 0.1, P < 0.005), detection of new follicles growing to > or = 5mm was negligible. Then, number of medium (4-5mm) growing follicles present in both ovaries decreased from 1.5+/- 0.3 at 0 h to 0.3+/- 0.1 at 72 h (P<0.005). In conclusion, the single ovulatory follicle is the largest growing follicle present in both ovaries at the moment of luteolysis. This follicle is selected to grow and ovulate while development of other follicles is inhibited.

Glucogenic supply increases ovulation rate by modifying follicle recruitment and subsequent development of preovulatory follicles without effects on ghrelin secretion

This study determined the effects of short-term energy inputs on ghrelin secretion and possible links with changes in the follicle population or the ovulation rate. Oestrous cycle was synchronized in 16 Manchega sheep using progestagen sponges and cloprostenol. Half of the animals were treated from days 0 to 4 by the oral administration, twice daily, of 200 ml of a glucogenic mixture containing 70% of glycerol, 20% of 1,2-propanediol and 10% of water; the control group received 200 ml water. The mean (+/-S.E.M.) plasma glucose increased immediately after the first administration (3.9+/-0.3 vs 3.0+/-0.1 mmol/l in control group, P<0.05), remaining statistically different during the treatment. However, plasma ghrelin levels were similar in both groups. On the other hand, the results indicated that short-term energy inputs modify ovulation rate (1.9+/-0.1 vs 1.3+/-0.2 in control group, P<0.05) by increasing the number of follicles able to be selected to ovulate during the period of treatment (>or=4 mm in size; 5.9+/-0.6 vs 4.3+/-0.4 at day 2, P<0.05). After sponge withdrawal, the number of these follicles decreased throughout follicular phase (5.8+/-0.8 to 1.5+/-0.4, P<0.0005) while the number of large follicles increased (>or=6 mm in size; 0.8+/-0.4 to 2.0+/-0.3, P<0.05); this would indicate an active growth of preovulatory follicles that were not found in the control group. Thus, the increases of ovulation rate by high-energy inputs would be caused by an enhancement in the developmental competence of preovulatory follicles.

Endogenous Circannual Cycles of Ovarian Activity and Changes in Prolactin and Melatonin Secretion in Wild and Domestic Female Sheep Maintained under a Long-Day Photoperiod

Abstract The present study examines the ovulatory activity of wild and domesticated ewes subjected to either a constant photoperiod of long days (16L:8D) or natural changes in daily photoperiod for 16 mo. The aim was to determine whether an endogenous reproductive rhythm controls seasonal reproductive activity in these sheep, and how the photoperiod might affect this. The effects of long-day photoperiods on long-term changes in prolactin and melatonin secretion were also evaluated. The two species showed changes in reproductive activity under the constant photoperiod conditions, suggesting the existence of an endogenous rhythm of reproduction. This rhythm was differently expressed in the two types of ewe (P < 0.05), with the domestic animals exhibiting much greater sensitivity to the effects of long days. A circannual rhythm of plasma prolactin concentration was also seen in both species and under both photoperiod conditions, although in both species the amplitude was always lower in the long-day animals (P < 0.01). The duration of the nocturnal melatonin plasma concentrations reflected the duration of darkness in both species and treatments. The peak melatonin concentration did not differ between seasons either under natural or long-day photoperiods.

Disruption of the endothelial nitric oxide synthase gene affects ovulation, fertilization and early embryo survival in a knockout mouse model

Two consecutive experiments determined whether disruption of the endothelial nitric oxide synthases (NOS) gene (Nos3) affects ovulation, fertilization, implantation, and embryo development. In the first trial, Nos3-knockout mice (groups Nos3(-/-)) and wild-type mice (groups Nos3(+/+)) showed significant differences in mean number of corpora lutea (9.7+/-1.2 in Nos3(-/-) versus 14.2+/-1.2 in Nos3(+/+); P<0.01), rate of anovulation (48.3+/-7.3% in Nos3(-/-) versus 29.7+/-6.3 in Nos3(+/+); P<0.05), total mean number of recovered oocytes/zygotes (4.0+/-1.1 in Nos3(-/-) versus 10.4+/-1.6 in Nos3(+/+); P<0.01), and non-fertilization rate (50.7 in Nos3(-/-) versus 3.3% in Nos3(+/+); P<0.001). In the second trial, implantation and early pregnancy losses in Nos3-knockout and wild-type dams were detected by real-time ultrasound imaging. The number of embryos reaching implantation was higher in Nos3(+/+) than in Nos3(-/-) mice (7.5+/-0.4 vs 4.0+/-0.4; P<0.005); thereafter, embryo losses were detected between days 8.5 and 13.5, in 62.5% of the Nos3-knockout dams and, at days 10.5 and 11.5, in 16.7% of the control females (P<0.005). Thus, NO and NOS3 deficiencies affect reproductive and developmental features in the Nos3-knockout mouse model.

Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance

The effects of undernutrition during pregnancy on prenatal and postnatal development of the offspring were evaluated in sows with obesity/leptin resistance. Females were fed, from day 35 of pregnancy onwards, a diet fulfilling either 100% (group control, n=10) or 50% of the nutritional requirements (group underfed, n=10). In the control group, maternal body weight increased during pregnancy (P<0.05) while it decreased or remained steady in the underfed group. At days 75 and 100 of gestation, plasma triglycerides were lower but urea levels were higher in restricted than in control sows (P<0.05 for both). Assessment of the offspring indicated that the trunk diameter was always smaller in the restricted group (P<0.01 at day 50, P<0.005 at days 75 and 100 and P<0.0001 at birth) while head measurements were similar through pregnancy, although smaller in the restricted than in the control group at birth (P<0.05). Newborns from restricted sows were also lighter than offspring from control females (P<0.01) and had higher incidence of growth retardation (P<0.01). Afterwards, during lactation, early postnatal growth in restricted piglets was modulated by gender. At weaning, males from restricted sows were still lighter than their control counterparts (P<0.05), while females from control and underfed sows were similar. Thus, the current study indicates a gender-related differential effect in the growth patterns of the piglets, with females from restricted sows evidencing catch-up growth to neutralise prenatal retardation and reaching similar development than control counterparts.

Relative roles of photoperiodic and nutritional cues in modulating ovarian activity in goats

The objective of the current study was to evaluate the influence of nutrition and its interaction with the photoperiod on the ovarian activity of Criollo goats. In early February (22 degrees NL, anestrous season) goats were randomly assigned to the two experimental groups: high (HN; n=10) and low (LN; n=10) nutrition goats. The HN group was fed in mixed prairies with grass and clover (17.3-/+7.5% of crude protein, CP; 66.3-/+5.7% dry organic matter, DOM) and received 150 g of concentrate (12% CP) per goat and day. The LN group received only corn stubble (6.2-/+0.7% CP, 53.7-/+1.9% DOM). Serum progesterone (P(4)) and triiodothyronine (T(3)) concentrations were measured (RIA) at three selected periods of seasonal anestrous: early (8-24(th) March), mid (13(th) April - 3(rd) May) and late (26(th) May - 14(th) June) anestrous. Body weight, body condition and body condition index were determined at the beginning of the study and every 14 days. Body weight was positively correlated with serum T(3) (r=0.704; p<0.05). The percentage of cycling does during the three examined periods was higher (p<0.05) in the HN group than in the LN group (80 vs. 30%, 80 vs. 20%, and 60 vs. 10%, respectively). The high nutrition level increased reproductive activity of Criollo goats during all three periods of the anestrous season including deep anestrous.

Influence of leptin on in vitro maturation and steroidogenic secretion of cumulus-oocyte complexes through JAK2/STAT3 and MEK 1/2 pathways in the rabbit model.

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus-oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.

Comparative Analysis of Muscle Transcriptome between Pig Genotypes Identifies Genes and Regulatory Mechanisms Associated to Growth, Fatness and Metabolism

Iberian ham production includes both purebred (IB) and Duroc-crossbred (IBxDU) Iberian pigs, which show important differences in meat quality and production traits, such as muscle growth and fatness. This experiment was conducted to investigate gene expression differences, transcriptional regulation and genetic polymorphisms that could be associated with the observed phenotypic differences between IB and IBxDU pigs. Nine IB and 10 IBxDU pigs were slaughtered at birth. Morphometric measures and blood samples were obtained and samples from Biceps femoris muscle were employed for compositional and transcriptome analysis by RNA-Seq technology. Phenotypic differences were evident at this early age, including greater body size and weight in IBxDU and greater Biceps femoris intramuscular fat and plasma cholesterol content in IB newborns. We detected 149 differentially expressed genes between IB and IBxDU neonates (p < 0.01 and Fold-Change > 1. 5). Several were related to adipose and muscle tissues development (DLK1, FGF21 or UBC). The functional interpretation of the transcriptomic differences revealed enrichment of functions and pathways related to lipid metabolism in IB and to cellular and muscle growth in IBxDU pigs. Protein catabolism, cholesterol biosynthesis and immune system were functions enriched in both genotypes. We identified transcription factors potentially affecting the observed gene expression differences. Some of them have known functions on adipogenesis (CEBPA, EGRs), lipid metabolism (PPARGC1B) and myogenesis (FOXOs, MEF2D, MYOD1), which suggest a key role in the meat quality differences existing between IB and IBxDU hams. We also identified several polymorphisms showing differential segregation between IB and IBxDU pigs. Among them, non-synonymous variants were detected in several transcription factors as PPARGC1B and TRIM63 genes, which could be associated to altered gene function. Taken together, these results provide information about candidate genes, metabolic pathways and genetic polymorphisms potentially involved in phenotypic differences between IB and IBxDU pigs associated to meat quality and production traits.