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We evaluated the effects of a constant low (5-5%) and modulated (5–20%) oxygen environments on the in vitro development of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) cultured in the presence or absence of an antioxidant (astaxanthin: Ax). OCGCs were cultured in a gas permeable culture device for 8 days in 5-5% O2 (±Ax) and 5–20% O2 (±Ax) culture conditions. In the oxygen modulated culture conditions, the oxygen concentration was switched from 5% to 20% on day 4 of culture. Ax promoted the viability of OCGCs (P 0.05). A constant low oxygen environment significantly promoted the blastocyst rates (P 0.05). In conclusion, exposure of OCGCs to constant low oxygen or oxygen modulation in the presence of Ax promotes the healthy development of OCGCs during the 8-day IVG culture using the gas permeable culture device.

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at −80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0–5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

The objective of this study was to characterize circulating Anti-Müllerian Hormone (AMH) concentrations in a population of Holstein heifers and examine the impact that life events and stage of life have on those concentrations. Virgin, Holstein heifers (n = 105) 13 ± 0.8 months old were heat detected using tail-chalk, bred via artificial insemination and pregnancy checked 32+ days later. Serum samples for AMH were collected upon enrollment (heifer), at 5-20 days in milk (fresh) and at 45-60 days in milk (pre-breeding). Transrectal ultrasonography was performed upon enrollment (heifer) and at 45-60 days in milk (pre-breeding) to determine antral follicle count (AFC), cyclicity status, and uterine health. Heifers were blocked into thirds by AMH concentration: HIGH (354 pg/mL; n = 34), MID (183-354 pg/mL; n = 35) and LOW (183 pg/mL; n = 36), with distribution re-evaluated at subsequent samplings (fresh, pre-breeding). As heifers, age and conception risk to first service were not impacted by AMH (P 0.05). Reason for leaving the herd, health incidences and calving difficulty were not impacted by AMH (P 0.05). AFC and cyclicity had a positive impact on heifer AMH (P 0.01). AFC and AMH in heifers were highly correlated (0.56, P 0.001). AFC for heifers differed by AMH group with the HIGH group having the greatest AFC (8.76), followed by the MID (5.87), then the LOW (3.53) group (P 0.0001). However, this association was not evident in the pre-breeding group (P 0.05). From the heifer to the fresh sample, average AMH dropped from 313.15 pg/mL to 160.01 pg/mL (P 0.0001). Average AMH at the pre-breeding sample was 183.23 pg/mL, which was lower than the heifer sample (P 0.0001), but not different from the fresh sample (P 0.05). AFC and AMH at the heifer sample had a positive impact on AMH at the fresh sample (P 0.01) and pre-breeding AMH was positively impacted by both the fresh and heifer AMH concentration (P 0.001). Most animals kept their AMH categorization through all three time points with more of the LOW AMH animals maintaining their categorization than the other groups. However, 32.1% of animals changed their AMH categorization from the heifer sample to the fresh sample, with 53.8% moving to a lower AMH categorization (corresponding to lower AMH) and 46.2% moving to a higher AMH categorization (corresponding to higher AMH). No differences were seen in circulating AMH based on health events however, differences in AMH concentration over time indicate a drop in circulating AMH post-calving. Circulating AMH concentration as a pre-breeding heifer is highly indicative of circulating AMH concentration as a first lactation animal and may be used to predict an adult animal's AMH concentration. However, it is necessary to compare AMH concentrations to herdmates as published AMH values vary widely from herd to herd. In addition, sampling time should be considered when determining AMH categorization of animals as circulating AMH concentration immediately post-calving may not be indicative of an animal's true AMH categorization.

The objective of this study was to evaluate the effects of reducing the sodium chloride content in in vitro growth (IVG) medium to 61.6 mM on in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium with 108 mM NaCl (αMEM-108) or porcine zygote medium (PZM) containing 61.6 mM (PZM-61.6) or 108 mM (PZM-108) NaCl. These media were further supplemented with 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 10% (v/v) fetal bovine serum. After IVG culture, oocytes were matured for 44 h in our standard IVM medium. The IVG culture in PZM-61.6 significantly increased nuclear maturation (88.0 ± 2.2%) of SAF oocytes compared to that in PZM-108 (77.3 ± 3.9%) or αMEM-108 (75.9 ± 3.8%). After parthenogenesis (PA), the proportions of blastocysts, based on the number of metaphase II (MII) oocytes, induced for PA were not different among IVG oocytes cultured in PZM-61.6 (50.2 ± 3.0%), PZM-108 (46.8 ± 2.9%), or αMEM-108 (45.6 ± 2.9%). The IVM oocytes derived from IVG in PZM-61.6 showed increased perivitelline space (PVS) (12.1 ± 0.6 μm) and intra-oocyte glutathione (GSH) content (1.19 ± 0.04 pixels/oocyte) compared to PVS (8.0 ± 0.5 and 7.4 ± 0.4 μm) and GSH (1.03 ± 0.04 and 1.00 ± 0.04 pixels/oocyte) of oocytes derived from PZM-108 and αMEM-108, respectively. The IVG culture in PZM-61.6 stimulated meiotic resumption after IVG and faster nuclear progression after IVM than that in αMEM-108. After somatic cell nuclear transfer (SCNT), the blastocyst formation of SAF oocytes grown in PZM-61.6 (17.8 ± 3.3%) was higher than that of oocytes grown in PZM-108 (7.5 ± 2.7%) but not different from that of oocytes in αMEM-108 (11.4 ± 3.4%). Regardless of the different osmotic pressures, nuclear maturation was significantly increased by IVG culture in PZM with reduced NaCl (86.8 ± 2.3 and 84.9 ± 4.2% in PZM-61.6 and PZM-61.6 with sorbitol, respectively) than in PZM-108 (70.5 ± 3.4%). Blastocyst formation was not affected by the differences in NaCl content and osmotic pressure of the IVG medium, whereas the mean number of cells in blastocysts was significantly higher following IVG culture in PZM-61.6 than in the other groups. In conclusion, the results demonstrate that, following SCNT in pigs, IVG culture of SAF oocytes in a medium with a reduced NaCl concentration stimulates oocyte maturation and improves subsequent embryonic development.

Letrozole is used for the treatment of subfertility in women undergoing ovarian superstimulation, but the mechanism of action has not been investigated critically. The objective was to test the hypothesis that treatment with letrozole will potentiate the superstimulatory response following gonadotropin treatment by increasing the number of follicles present at ovarian follicular wave emergence in cattle. In Experiment 1, ovarian follicular wave emergence was synchronized among beef heifers (n = 8) by transvaginal ultrasound-guided follicle ablation. On Day 0 (wave emergence), a letrozole-releasing device (LRD) was placed intravaginally for 5 days, followed again by transvaginal follicle ablation on Day 5. The number of follicles ≥3 mm was recorded by transrectal ultrasonography on Days 0 and 6.5 (i.e., pre- vs. post-LRD treatment). In Experiment 2, non-lactating dairy cows were assigned randomly to one of two groups (n = 15/gp) after follicle ablation-induced synchronization of wave emergence (Day 0), and given either an LRD or sham device for 5 days. Superstimulatory treatment was initiated on Day 0, consisting of 8 doses of 50 mg of porcine FSH im at 12 h intervals, and luteolytic doses of prostaglandin on Days 3 and 3.5. The LRD/sham devices were removed on Day 3.5, GnRH was given im on Day 5, estrus response was determined on Days 5 and 6, and the ovarian response was recorded by ultrasonography on Days 0, 3.5, 5, 6.5, and 12. In Experiment 1, no difference was detected in the number of antral follicles at wave emergence pre- vs. post-LRD treatment (23.2 ± 3.2 vs. 23.5 ± 3.8 follicles; P = 0.67; mean ± SEM). In Experiment 2, the interval from prostaglandin treatment to estrus was longer (50.3 ± 1.1 vs. 40.7 ± 2.0 h; P 0.001) and less variable (residuals: 3.1 ± 0.5 vs. 6.7 ± 0.9 h; P 0.01) in the LRD vs. sham group. The proportion of ovulations (number of CL on Day 12 over the number of follicles ≥3 mm on Day 0) did not differ (0.65 ± 0.02 vs. 0.70 ± 0.02; P = 0.15) nor did the number of CL on Day 12 (15.9 ± 2.5 vs. 19.0 ± 2.0; P = 0.32) between the LRD and sham groups. In summary, treatment with letrozole did not increase the number of antral follicles at wave emergence or the superstimulatory response, but increased precision in the interval to estrus and may be useful for artificial insemination at a fixed time in superstimulatory protocols.

Made available in DSpace on 2020-12-12T01:34:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) C-type natriuretic peptide (CNP) and its natriuretic peptide receptors subtype 2 (NPR2) are essential for the maintenance of oocyte meiotic arrest in different species. Extracellular vesicles (EVs) in bovine follicular fluid (FF) are important for cell communication within the ovarian follicle. This study investigated the involvement of EVs from FF of bovine ovarian follicles in the CNP-NPR2 system, first by analyzing the presence of CNP in the EV contents, followed by addition of EVs to in-vitro maturation (IVM) medium, to evaluate the effect on maintenance of oocyte meiosis arrest and improvements in in-vitro embryo production. As expected, CNP was observed in FF and granulosa cells from the ovarian follicles. To the best of our knowledge, this is the first time that CNP has been found in the EV contents. To evaluate the possible effect of EVs on the progression of oocyte meiosis, the IVM was performed under three conditions: CNP and EV supplementation and control condition. Both the CNP and EV treatments inhibited meiosis resumption in the oocyte within 9 h of IVM. CNP treatment increased cGMP levels in cumulus cells within 6 h of IVM compared to the control group, but the EV treatment did not. In contrast, the relative mRNA abundance of adenylate cyclase 3 and 9 (ADCY3 and ADCY9) was upregulated in oocytes after 6 h of IVM under EV treatment compared to the control group, but not under CNP treatment. Last, these treatments in the IVM medium had no significant effect on the in-vitro embryo production. In conclusion, we demonstrated the presence of endogenous CNP in bovine reproductive structures, especially in the EVs from the FF of antral follicles. The presence of CNP in the EVs suggests an important involvement of this cell-communication system in the CNP-NPR2 system. Therefore, we indeed observed that the EVs from FF can modulate the arrest of oocyte meiosis, acting similarly to the CNP-NPR2 system to block the oocyte in the GV state. However, the mechanism of each system might be different; the CNP-NPR2 system seems to be involved in modulating the cGMP levels, while the contents of EVs might be involved in modulating the cAMP levels. São Paulo State University (UNESP) Institute of Biosciences Department of Pharmacology University of São Paulo (USP) Faculty of Animal Science and Food Engineering Department of Veterinary Medicine São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Sciences São Paulo State University (UNESP) Institute of Biosciences Department of Pharmacology São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Sciences FAPESP: 2012/50533-2 FAPESP: 2013/07730-4

Oocyte pick up (OPU) coupled with IVP produce over 1 million cattle embryos per year and has been most successful in Bos indicus derived breeds that contain large numbers of antral follicles on their ovaries. More recently, this technology has been applied on a large scale to Bos taurus cattle, where hormone manipulation is generally employed to improve the developmental competence of the COCs. Hormone manipulation, and specifically the use of FSH priming before OPU, has been strategically used in the intensively managed dairy cow, where genomic evaluation and juvenile IVP can produce additional significant genetic gains.

The objective of ovarian superstimulatory treatments in cattle is to obtain the maximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes. While factors inherent to the donor animal are critical, an increased knowledge of ovarian physiology, gonadotropin biochemistry and the ability to manipulate ovarian function have provided alternatives for the design of simple and successful protocols for superovulation in cattle. Recent protocols have also been made more user-friendly and allowed for the grouping of donors for successful superovulation. Although the number of reports associating FSH profiles with superovulatory response is limited, studies designed to reduce the number of FSH treatments necessary to induce superstimulation may provide guidance for the development of optimized gonadotropin treatment protocols. Although high peak levels of circulating FSH following a single administration of Folltropin-V have been shown to be associated with a reduced superstimulatory response, the ideal treatment protocol would seem to be to increase circulating FSH levels to values comparable to those required for the induction of follicle wave emergence, and to maintain these levels for at least 72 h (or 36 h for superstimulation prior to ovum pick-up) to allow follicles to reach an ovulatory size and acquire the capacity to ovulate.

The use of assisted reproductive technologies, such as superovulation and in vivo embryo production and in vitro embryo production (IVEP), has increased rapidly in recent years and is now applied worldwide for genetic improvement in beef and dairy buffaloes. Although in vivo embryo production has been shown to be feasible in buffalo, low efficiency and limited commercial application has been documented. These results could be associated with low antral follicle populations, high levels of follicular atresia and/or failures of the oocyte to enter the oviduct after superovulation. Additionally, IVEP technology has been shown to be an important tool for multiplying genetic material from donors of superior merit, and promising results have been achieved with the use of ovum pick-up (OPU) along with IVEP in buffalo. However, several factors appear to be critical for successful OPU/IVEP, including circulating levels of anti-Müllerian hormone, antral follicle populations, sizes of the follicles available for the OPU, reproductive seasonality, semen (sire) used for IVEP, donor category and farm. Furthermore, technologies applied to control follicular wave emergence and ovulation at predetermined times, without the need for estrus detection in recipients, has facilitated management and improved the efficiency of embryo transfer programs in buffalo herds. Conclusively, with the considerable evidence of poor results with in vivo embryo production in buffaloes, the association of OPU with IVEP represents a new alternative for the exploitation of buffalo genetics.

Granulosa cells (GCs) contribute to oocyte development. The present study addressed the effect of cryopreservation on the ability of GCs to support oocyte development. GCs were collected from antral follicles. Oocyte granulosa cell complexes (OGCs) derived from early antral follicles were cultured with additional fresh-GCs or frozen-thawed-GCs for 14 days, and the developmental ability and characteristics of the oocytes grown in vitro were examined. Furthermore, fresh- or frozen-thawed-GCs were cultured for two days, and the effects of cryopreservation on the characteristics of GCs were examined. The developmental ability of blastocysts and the acetylation levels of H4K12 in oocytes grown in vitro did not significantly differ among the three culture conditions: OGCs cultured with additional fresh-GCs, frozen-thawed-GCs, or without additional GCs. Although both fresh- and frozen-thawed-GCs exhibited increased ATP content compared with that in oocytes developed without additional GCs, only fresh-GCs showed significantly increased lipid content in oocytes grown in vitro. ATP content, reactive oxygen content, mitochondrial membrane potential, and mitochondrial DNA copy number were greater in cultured frozen-thawed-GCs compared with fresh-GCs. In contrast, lipid content of cultured frozen-thawed-GCs was lower than that of fresh-GCs. Both fresh- and frozen-GCs support oocyte growth, but cryopreservation changes the properties of GCs in a manner that affects the energy status of oocytes grown in vitro.

Stem cell factor (SCF) plays important roles in primordial follicle activation, oocyte growth and survival, granulosa cell proliferation, theca cell recruitment, and ovarian steroidogenesis. The aim of this study was to investigate the effect of SCF on in vitro growth of buffalo oocytes. Oocyte-granulosa cell complexes (OGCs) were dissected from early antral follicles of slaughtered buffalo ovaries and cultured for 6 days in media supplemented with 0, 50 or 100 ng/mL SCF. In vitro grown oocytes were further cultured for in vitro maturation for 24 h. The results showed that SCF significantly (P 0.05) increased oocyte diameter in vitro. The percentages of surviving oocytes were 60, 81 and 92 in 0, 50 and 100 ng/mL SCF supplemented group, respectively. SCF promoted formation of antrum-like structures in culture. The results also showed that SCF enhanced the maturation of in vitro grown buffalo oocytes. Here, 14% in vitro grown oocytes reached metaphase II (MII) stage in 50 ng/mL SCF supplemented group, whereas the percentage was increased to 26% in 100 ng/mL SCF treated group. These results show that SCF supports the growth, viability and nuclear maturation of buffalo oocytes in vitro.

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p

An experiment was performed to evaluate the association between the antral follicle count (AFC) plus body condition score (BCS) and the pregnancy rate in Bos indicus undergoing timed artificial insemination (TAI). A total of 736 Nelore cows with BCSs ranging from 2 to 4 received a conventional protocol for TAI. On a random day of the estrous cycle (Day 0), all cows received an intravaginal P4 device and an intramuscular (i.m.) injection of 2.0 mg estradiol benzoate. On Day 8, the P4 device was removed, and 150 μg sodium D-cloprostenol, 300 IU equine chorionic gonadotrophin and 1.0 mg estradiol cypionate were administered by i.m. injection. TAI was performed 48 h after P4 device removal, and pregnancy diagnosis was performed by ultrasonography after 30 days. On Day 0, all cows were examined by ultrasonography to determine the AFC by counting the number of follicles >3 mm in diameter that were present in both ovaries and to evaluate the BCS (scale of 1–5). The cows were then classified based on their AFCs as those with low (≤10 follicles), intermediate (11–29 follicles) and high AFC (≥30 follicles). Furthermore, cows were classified as having low (≥2.0 to ≤ 2.9) and high (≥3.0 to ≤ 4.0) BCSs. The AFCs and BCSs were analyzed using the generalized linear model, and the pregnancy rate was assessed with the binary logistic regression model (P ≤ 0.05). The pregnancy rate was influenced (P 30 and a BCS between 3 and 4. In conclusion, AFC and BCS classifications influence the pregnancy rate of Bos indicus beef cattle subjected to TAI. In addition, an important interaction between these factors was observed, namely, the lowest pregnancy rates were found in cows with high BCSs and high AFCs.

The objective of the present study was to examine the effects of neonatal exposure to either agonists or antagonists of androgen and estrogen receptors on the expression of growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) and their cognate receptors (TGFBR1, BMPR1B, and BMPR2) in ovarian follicles of adult pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen, at 20 mg/kg bw), flutamide (FLU, an antiandrogen, at 50 mg/kg bw), 4-tert-octylphenol (OP, an estrogenic compound, 100 mg/kg bw), ICI 182,780 (ICI, an antiestrogen, 400 μg/kg bw), or corn oil (control) between postnatal Days 1 and 10 (n = 5/group). Ovarian follicles were excised from adult pigs on Days 8–11 of the estrous cycle. The expression of GDF9, BMP15, TGFBR1, BMPR1B and BMPR2 were examined in the population of preantral and small antral ovarian follicles using real-time PCR, Western blot and immunohistochemistry. In preantral follicles, the upregulation of GDF9 mRNA and protein expression was found in pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMP15. TGFBR1 and BMPR2 mRNA and protein expression were upregulated in preantral follicles of adult pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMPR1B. In small antral follicles, the mRNA and protein for TGFBR1 and BMPR2 were upregulated, while BMPR1B was downregulated in response to neonatal OP treatment. In addition, treatment with FLU upregulated BMPR1B and BMPR2 mRNA and protein expression, while downregulated the expression of TGFBR1. Moreover, GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles obtained from both control and treated ovaries. TGFBR1, BMPR1B and BMPR2 receptors were observed in the oocytes and granulosa cells of preantral follicles as well as in granulosa and theca cells of small antral follicles. In conclusion, the present study demonstrated neonatal exposure to either agonists or antagonists of androgen and estrogen receptors affected GDF9 and BMP15 signalling in ovaries of adult pigs. It seems that neonatal androgen excess or deficiency may lead to the acceleration of initial follicle recruitment, while neonatal exposure to compounds with antiandrogenic and estrogenic activity may disturb small antral follicles fate. Therefore, it confirms that neonatal window is critical for programming of ovarian function in pigs.

Anti-Mullerian Hormone (AMH) has been linked to reproductive tract abnormalities in mares and stallions. This study aimed at evaluating AMH as a biomarker for two reproductive conditions in mares. In the first part of this study, plasma AMH was evaluated as an early indicator of the onset of cyclicity in mares in the transitional period from the anovulatory phase during winter anoestrus to the cyclic phase during the breeding season. Ten mares between 8 and 17 years old were included in the experiment which lasted from mid-February until the end of April. Ovarian activity was monitored with ultrasonography three times per week, the detection of a corpus haemorrhagicum/luteum was documented and antral follicle counts (AFC) were recorded. Blood samples were collected weekly by jugular venipuncture during the whole study period to compare AMH concentrations before and after the first ovulation of the year. The second objective was to evaluate if plasma AMH concentrations in middle-aged mares are linked to fertility and could serve as a prognostic marker in that age group. A total of 41 privately-owned clinically sound mares aged between 12 and 21 years of various breeds were inseminated with fresh or frozen semen. Mares were scanned between day 14 and 20 and the “early pregnancy rate” included only positive pregnancy examinations after the first observed cycle in the season of each mare. Potential associations between the early pregnancy rate in the first cycle and the explanatory factors AMH concentrations, age, status of the mare, stud, development of post-breeding endometritis, number of inseminations and semen type were analysed using logistic regression models. In the first part of the study, correlation between AMH and AFC for the whole study period (P = 0.0002, ρ = 0.55) as well as prior to (P = 0.008, ρ = 0.58) and after the first ovulation (P = 0.0007, ρ = 0.69) were observed. However, AMH concentrations before and after the first ovulation of the year were not statistically different. The second part of the study revealed no association between early pregnancy rate and AMH concentrations or any of the other mentioned factors. In conclusion, this study showed no evidence of a difference between AMH concentrations before and after the first ovulation of the year thus not supporting the use of AMH as a biomarker to predict the onset of cyclicity in mares. We could furthermore not show a relationship between plasma AMH concentrations and early pregnancy rates in this cohort of animals.

The aim of this study was to evaluate the effect of active immunization against GnRH in Nelore (Bos indicus) cows repeatedly used as oocyte donors that developed chronic cystic ovarian disease (COD). In the first experiment, ovarian and uterine features were first compared between COD cows (n = 15) and healthy cows (n = 22, cycling control group) from the same breed and herd. Cows with COD had a greater number of large (P 0.0001) and medium follicles (P 0.01) but lesser small follicles (P 0.05) than cycling controls. Mucometra was diagnosed in 73.3% of COD cows, but in none of the controls. No difference in average thickness of the endometrium was detected between groups; however, endometrial thickness and mucometra score were negatively correlated (R = -0.73, P = 0.0029) in COD cows. In the second experiment, COD cows were randomly allocated into two experimental groups, which received two 1.0 mL SC injections of either an anti-GnRH vaccine (COD immunized group, n = 8) or saline (COD control group, n = 7), given 28 days apart. Cows were examined weekly by transrectal ultrasonography during nine consecutive weeks after the first injection to evaluate the number and distribution of follicles among size classes, endometrial thickness, and presence of clinical mucometra. Vaccination against GnRH resulted in a progressive suppression of follicle growth and a reduction in the average size of the largest follicle, as well as in the number of large follicles (P 0.01) in COD immunized cows compared with COD controls from week 7 onwards. Conversely, the number of small follicles in the COD immunized group increased after week 5 and was greater (P = 0.0023) than controls on week 9. Endometrial thickness and mucometra score were not affected (P 0.05) by immunization against GnRH. In the third experiment, the COD immunized cows with effective suppression of follicle growth four weeks after the 2nd injection (n = 6) were submitted to three consecutive ovum pick-up (OPU) sessions (weeks 10, 11, and 12) for in vitro embryo production (IVEP). Cumulus-oocyte complexes (COC) collected from slaughterhouse ovaries were used as controls for IVEP. COD cows with produced 25.0 ± 3.8 COC per OPU session with no apparent detrimental effect of anti-GnRH vaccine on oocyte developmental potential in vitro, i.e., we observed similar cleavage rate (P = 0.5914) and greater blastocyst rate (P = 0.0177) in immunized cows compared with COC from slaughterhouse controls. Finally, in the fourth experiment wave emergence and follicular dynamics after follicle ablation were compared between COD immunized cows with effective suppression of follicle growth and a subset (n = 6) of the cycling, control group. No follicles grew beyond 4 mm diameter after follicle ablation in the COD immunized group, whereas a normal follicular wave emergence occurred in cycling controls. Antral follicle count was similar between cycling controls and COD immunized groups at 24 h and 96 h post-follicle ablation (P 0.05), but greater in cycling controls at 48 h and 72 h post-follicle ablation (P 0.05). In summary, our results suggest that active immunization against GnRH is effective to induce the regression of follicular cysts as well as increase the number of small follicles growing on the ovaries, in oocyte donors diagnosed with chronic COD, with no apparent negative effect on oocyte developmental potential in vitro.

Information on the follicular population and oocyte quality of cows in the final period of reproductive life is scarce. The present study aimed to compare the antral follicle count (AFC), oocyte production and embryonic developmental competence of young versus long-lived and senescent Bos indicus beef cows. Nellore cows (Bos indicus) were classified into three groups according to age: young (4-9 years, n = 10), long-lived (14-17 years, n = 10) and senescent (17-23 years, n = 10). At a random time in the estrus cycle, the cows received cloprostenol sodium salt (0.5 mg, IM), estradiol benzoate (1 mg, IM) and an intravaginal P4 device (1.4 g). Five days later, the P4 devise was removed and oocyte collection (OPU1) was performed. A second OPU (OPU2) was performed 5 days after the first in order to aspirate only growing follicles. During each OPU, AFC and the number and quality of cumulus-oocyte complexes (COCs) were evaluated. Then, the COCs were placed in standard maturation medium (IVM), fertilized and incubated for 9 days. The data were subjected to ANOVA and Multinomial Logistic Regression. The AFC was smaller in long-lived and senescent cows in both OPU1 and OPU2 when compared to younger cows. There was no difference in AFC between OPU1 (19.9 ± 1.8) and OPU2 (17.6 ± 1.9) in young cows, however, more follicles were punctured in long-lived and senescent cows in OPU1 (12.0 ± 2.6 and 19.3 ± 4.6) than in OPU2 (9.2 ± 1.9 and 10.3 ± 2.3), respectively (P 0.01). The numbers of COCs recovered from young cows (OPU1 = 14.2 ± 1.8; OPU2 = 8.4 ± 0.9) were higher than those obtained from long-lived cows (OPU1 = 5.9 ± 2.3; OPU2 = 4.3 ± 1.0) and senescent cows (OPU1 = 7.2 ± 3.0; OPU2 = 4.1 ± 1.7), respectively (P 0.05). The cleavage rate did not differ between groups. However, the rate of blastocyst formation was higher for young (64.8%) and long-lived (65.0%) compared to senescent (16.5%) cows (P 0.01). In conclusion our results indicate that the AFC is lower in long-lived and senescent cows compared with young cows. However, unlike in senescent cows, the embryonic development of long-lived cows is similar to that of young cows. This suggests that Nellore cows aged17 years begin to have reduced embryonic development capacity due to ovarian aging.

Oocytes in vivo generally mature in ovarian follicles that are soft, whereas oocytes that mature in vitro are on the hard surface of culture dishes. Embryonic ontogeny through organogenesis has greater ability in in vivo matured oocytes than it does in in vitro matured oocytes, indicating the importance of a soft culture matrix. In this study, we report the effect of using an agarose matrix as a culture substrate on the development of pig oocytes derived from medium antral follicles. The cumulus-oocyte complexes (COCs) retrieved from medium antral follicles were matured on noncoated (control) culture dishes or dishes coated with 1% and 2% (w/v) agarose matrices. Subsequently, the effect of the soft culture matrix on the developmental competence of porcine oocytes was assessed by analyzing cumulus expansion, blastocyst formation after parthenogenetic activation (PA), gene expression levels (ACTN4, BMP15, BAX, HIF1A, PFKP and VEGFA), TUNEL indices, BMP15 protein expression levels, cortical granule (CG) distribution, and intraoocyte ATP levels. In vitro maturation (IVM) of pig COCs using a 1% (w/v) agarose matrix resulted in significantly higher blastocyst formation, cumulus expansion, gene expression of BMP15, HIF1A and VEGFA, protein expression of BMP15, and intraoocyte ATP levels, and there was significantly reduced expression of a pro-apoptotic gene and ACTN4 gene and a reduction in TUNEL indices. These results demonstrate that the developmental competence of porcine oocytes can be effectively improved through IVM on a soft culture matrix made of agarose over what is observed using hard culture dishes.

In vitro growth (IVG) culture of bovine oocyte-cumulus-granulosa complexes (OCGCs) is generally carried out for 12 or 14 days using conventional gas impermeable culture devices. The culture duration may be longer compared to follicular development in vivo. During follicular development, follicles receive oxygen from micro vessels; however, oxygen supply is limited under the culture using conventional gas impermeable devices. The purpose of this study was to investigate the effect of increasing dissolved oxygen availability using a gas permeable (GP) culture device with or without antioxidant (astaxanthin, Ax) supplementation on 8-day IVG culture systems for bovine OCGCs derived from early antral follicles. We cultured OCGCs in GP, GP supplemented with Ax (GP + Ax), and a conventional gas impermeable device (control) for 8 or 12 days. OCGC viability were significantly higher when cultured for 8 days than 12 days (p 0.001) in all culture condition, but significant difference was not observed between groups (p 0.05). Antrum formation rates of OCGCs were higher after 12 days than 8 days of culture in all culture condition (p 0.001) and were significantly higher in the control than GP groups regardless of Ax supplementation (p 0.05). Oocyte diameters were similar among day-8 GP + Ax, day-8 control and day-12 control groups (p 0.05). Nuclear maturation rates of oocytes grown in vitro for 8 days were significantly higher in the GP + Ax group than in the control and the GP groups (p 0.05) and similar to oocytes grown for 12 days regardless of the culture conditions (p 0.05). The generation of reactive oxygen species in OCGCs on day 8 of IVG culture was significantly lower in the GP + Ax group than those of the GP and control groups (p 0.05). IVG oocytes after eight days of culture developed into blastocysts, and the cleavage and blastocyst rates were similar in all treatment groups. However, in vivo-grown oocytes had significantly higher (p 0.05) cleavage and blastocyst rates than the IVG oocytes in all groups. The present study demonstrates that increased oxygen availability using a GP culture device with Ax supplementation promotes oocyte growth and maturation competence but inhibits proliferation of granulosa cells and antrum formation compared with a conventional gas impermeable culture device, and that OCGCs can attain developmental competence after 8 days of IVG culture.

Among the reproductive biotechnologies, in vitro embryo production (IVEP) is an important tool for multiplying genetic material of superior merit. Recently, the number of embryos produced and transferred in vitro became significantly higher than that produced in vivo worldwide. In this context, the enhancement was attributable to ovum pick-up (OPU). With the advent of genomic technology, shortened breeding intervals, and increased selection accuracy, IVEP has attracted increasing attention for commercial use. The IVEP technique is well-established, but the embryo production rate has reached a plateau at 30–40%. Despite constant advances, the OPU/IVEP programs face some challenges that hinder the efficient application of the technique. Previous studies have shown that the quantity and quality of aspirated oocytes are essential factors for successful IVEP. This paper presents a brief overview of alternatives that can be employed to improve the process-seeking methods that assist in the recovery of better-quality oocytes and higher competence in OPU to improve embryo production. These strategies include using follicular wave synchronization prior to OPU, employing the influence of antral follicle populations, using the pre-OPU gonadotrophic stimulus and applying non-hormonal methods for selecting female donors.

To determine relationships of ovarian antral follicle count (AFC) on d 28 postpartum with postpartum interval (PPI) and AFC on d 8 post-estrus, three-dimensional ultrasonography was performed on multiparous (n = 47) and primiparous (n = 20) Brahman cows on d 28 postpartum and on d 8 post-estrus. Follicles were evaluated to determine numbers of total, small (≤17 mm3), medium (17 mm3–67 mm3), and large follicles (≥67 mm3) on each ovary. Estrus was detected using vasectomized, penile-deviated bulls fitted with chin-ball markers during the AI period and with tail paint during the natural service period. Cows with AFC ≥ ½ SD above the mean were classified as high, those within ½ SD of the mean were classified as intermediate, and those ≥ ½ SD below the mean were classified as low. Classes of AFC for total follicles included low (L ≤ 73), intermediate (74 ≤ I ≤ 112), and high (H ≥ 113). Classes of AFC for small follicles included low (L ≤ 55), intermediate (56 ≤ I ≤ 87), and high (H ≥ 88). Classes of AFC for medium follicles included low (L ≤ 13), intermediate (14 ≤ I ≤ 23), and high (H ≥ 24). Classes of AFC for large follicles included low (L ≤ 2), intermediate (3 ≤ I ≤ 4), and high (H ≥ 5). Cows with PPI ≥ ½ SD above the mean were classified as long, those within ½ SD of the mean were classified as intermediate, and those ≥ ½ SD below the mean were classified as short. Classes of PPI included short (S ≤ 47 d), intermediate (48 d ≤ I ≤ 75 d), and long (L ≥ 76 d). The PROC MIXED procedure of SAS was used to analyze potential differences among classes of AFC and PPI. The PROC REG procedure of SAS was used to analyze relationships between AFC and PPI and between AFC on d 28 postpartum and on d 8 post-estrus. The PPI differed among classes of total (P

Anti-Müllerian hormone (AMH) has gained increasing interest as a biomarker for assessment of gonadal activity. The ability to predict the ovarian follicular reserve of prepubertal female horses (fillies) or to identify stallions with testicular pathologies already during their prepubertal life has not been analyzed so far. Both would help to select fertile horses and reduce costs associated with keeping animals. The objectives of the present study were to (1) assess AMH, LH, FSH, progesterone (females) and testosterone (males) dynamics in prepubertal horses from birth onwards and (2) determine whether AMH concentrations detected in plasma of prepubertal female and male horses are correlated with postpubertal gonadal development. Warmblood foals (n = 30, 14 females, 10 normal males and 6 males with abnormal testicular development) born between February and May of two consecutive years (n = 28 in the first year and n = 2 the next year), were included in the study. Information on gestational length, parity of the dam and placental weight was collected for all foals. Blood samples for hormone analysis were collected from birth onwards every four weeks up to the age of one year. At two years, blood samples were collected on the day when antral follicle count (AFC) and total testicular volume (TTV) were assessed. AMH was detectable in the plasma of all animals from birth onwards and its concentration was significantly higher (P .001) in males than in females, regardless of testicular development. In males, AMH and testosterone concentration were similar for all animals during the first year of life, regardless of testicular development. At two years, AMH concentration was higher (P .05) in males with abnormal testicular development than in those with normal testes. In females, AMH concentration at two years was correlated with AMH concentration at birth (P .05) and with AFC (P .001). At birth, LH concentration was lower (P .05) in stallions with abnormal testes (0.3 ± 0.2 ng/ml) than in controls (0.6 ± 0.2 ng/ml). A high negative correlation between AMH concentration and gestation length was observed in males during the first eight weeks of life (P .01, r = -0.64 to -0.71). Elevated progesterone concentrations over 1 ng/ml were observed in several females starting with 20 weeks of age. This was paralleled by an increase in AMH concentration and was preceded by FSH and LH increases. In conclusion, AMH determination can be reliably used from two years onwards to identify stallions with abnormal testicular development, but it is inconclusive before puberty. In female horses, determination of AMH concentration at a prepubertal age allows for prediction of AMH and AFC after puberty. We suggest that premature luteinisation occurs before the onset of puberty in female horses and that LH secretion in the perinatal period is involved in testicular development and descent in the horse.

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis.

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was to estimate the pleiotropic effects of these carcass quality oriented markers for body weight, reproduction, and first calf performance traits in 174 replacement beef females which were managed under 2 post-weaning development protocols. Heifers were weighed at 11-, 12-, and 13-mo, at first breeding season pregnancy evaluation, and prior to first calving season. Pubertal status was determined at 11-, 12-, and 13-mo of age. Antral follicles were counted, reproductive tracts were scored, and tract dimensions were measured at 13-mo. Body condition and hip height were scored and measured at pregnancy evaluation and prior to calving season. Heifer pregnancy and weaning rates and ordinal birth date were recorded. Calf body weights at birth and weaning were analyzed. Single df linear contrasts for recessive effects of the GHR heterozygous genotype showed significant decreases of 2.5–3.6% in 11-, 12-, and 13-mo heifer body weights and heifer weight prior to calving. The additive differences between GHR homozygotes were small and not significant for all body weights measured but a 1 wk difference in calf birth date was significant. For all 13-mo uterine measurements, scores, and antral follicle counts, only the CAST dominance contrast for medium antral follicle count was significant. The CAPN1 haplotype with a strong additive effect for increased beef tenderness also had a significant additive effect on calving date. Heifers homozygous for the tender haplotype calved 7.9 days later than heifers homozygous for the tough haplotype. Most heifer reproductive traits were not significantly affected by CAST and CAPN1 markers that are widely used to improve beef tenderness by selection and breeders should not be concerned with how these markers affect reproduction and other heifer traits with the possible exception of CAPN1 effects on calving date.

Research on reproduction and fertility is an essential fundament of management strategies in European wild boar. Concerning the examination of the reproductive tract, methods still lack conformity and comparability. So far the reproductive status of female wild boar has been assessed by ovarian macroscopic examination. In order to find out if this is a valid method, 21 ovaries of 8–34 months old animals covering (early) follicular phase and cyclic inactivity were evaluated microscopically. Number and dimension of follicles as well as the occurrence of atretic follicles were compared and related to age, weight, ovary weight and size of ovary. For this study, 21 ovaries of the right side of each animal were weighed and measured. The diameter of the functional bodies was measured and functional bodies were counted. Size of ovary was calculated by length*width*height (cm3). Macroscopically, follicles were classified into four categories by diameter: F1 (0.2–0.3 cm), F2 (0.4–0.5 cm), F3 (0.6–0.8 cm) and F4 (0.9–1.3 cm). Microscopically, antral follicles of every 10th slice per section of each ovary were counted and classified into three categories by diameter: F0 (≥2 mm), FS (1–2 mm) and FXS (≤1 mm). In follicles with a diameter greater than 2 mm (F0), length and width were measured from the beginning of theca interna. Results showed that age and weight of wild boars were highly correlated (cor = 0.845, p

This study aims to compare four different methods for selecting high responding sheep donors for in vivo embryo production. These methods include a pre-selection eCG test (eCG), antral follicle count (AFC), plasma anti-Mullerian hormone measurement (AMH) and genotyping for the presence of the FecGE mutation (a polymorphism in the GDF9 gene associated with increased ovulation rate). Santa Ines ewe lambs (n = 25) underwent superovulation (SOV) with 800 IU equine chorionic gonadotropin (eCG) and the corpus luteum (CL) count was recorded by laparoscopy after eight days. At the D0eCG, blood samples for AMH and genotyping analysis were collected. Twenty-one days after the end of the eCG test, the same animals underwent SOV with 200 mg of FSH, administered in six decreasing doses, and then naturally mated. Immediately before the beginning of the FSH protocol (D0FSH), and at the moment of the first FSH dose (D9FSH), the AFC was assessed. Plasma AMH was again determined at the D9FSH. After each screening process, animals were classified as having a high (HR), or low (LR), potential of response (using specific thresholds for each method). Then, the ewes' response to SOV and embryo yield for each screening method, classified as HR or LR, were compared. Animals classified as HR by AFC (HRAFC) and by AMH concentration (HRAMH) at the D9FSH, produced more viable embryos than those classified as LRAFC and LRAMH (HRAFC 6.2 ± 3.2 vs LRAFC 2.8 ± 3.0 and HRAMH 6.6 ± 3.6 vs LRAMH 3.0 ± 2.9). Pre-selection tests with eCG and different FecGE genotypes, either heterozygous (+/E) or wild type (+/+), were unable to discriminate HR or LR animals. A tendency (P = 0.06) to have lower plasma AMH was observed in heterozygous FecGE (+/E) ewes. In conclusion, both AFC and plasma AMH can be used to select donor ewes with a higher potential of response for in vivo embryo production.

International audience; Ovarian follicular growth occurs in both the follicular and luteal phases of the estrous cycle but in very different endocrine contexts. In both phases, many small antral follicles with similar morphologic and histologic characteristics are present within the ovaries as a reserve for the terminal folliculogenesis. However, there are several gaps in our molecular knowledge of the gene expression profiles of small antral follicles in the follicular and luteal phases. The aim of the present study was to use RNA sequencing to compare and analyze the global transcriptional profile of ovine granulosa cells collected from small antral follicles (1-3 mm) either during the follicular or the luteal phase of the estrous cycle, with the hypothesis that they should be differential. We identified 663 genes whose mRNA was differentially expressed or accumulated in the granulosa cell layer of small antral follicles in the two phases. A comprehensive interpretation of these data was performed through integrative analyses (Gene Ontology, Ingenuity Pathway Analysis) and the exploitation of already available transcriptomic data on follicular growth and atresia. In particular, we observed that the contrasted endocrine context between follicular and luteal phases may have an impact on estradiol, follicle-stimulating hormone (FSH), and on the activin/inhibin signaling pathways. Furthermore, we reveal the possible initiation of early follicular atresia in small antral follicles during the follicular phase in interaction with the presence of immune cells. This study provides new insights into the gene expression profile in ovine granulosa cells, and we suggest that these molecular changes may have an implication at the time of follicle selection. (C) 2017 Elsevier Inc. All rights reserved.

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.

Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo. The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5–1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17β (E2), but less progesterone (P4). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P

This study was conducted to evaluate the effects of cyclic AMP (cAMP) modulator cilostamide (CIL) and forskolin (FSK) treatment during in vitro growth (IVG) on growth, maturation, and embryonic development of cumulus-oocyte complexes (COCs) derived from small antral follicles

Follicular growth and ovulation of healthy oocytes is a complicated process which is regulated by several endocrine and paracrine factors as well as cross-talk between the oocyte and its surrounding somatic cells. This study compared the expression profile of some candidate genes involved in BMP signaling as well as estrogen and AMPK production in cumulus-oocyte complex (COC) of small and large antral follicles and their associated somatic cell layers in ovaries from ewes with high- and low-antral follicle count (AFC). Expression of GDF9 was increased by increasing the size of antral follicles, while BMP15 expression was decreased by follicular size. It should be noteworthy that transcription of both GDF9 and BMP15 was also detected in the adjacent cellular layers under the follicles. There was a very strong positive correlation between BMP15 and BMPR2 in ovary tissues. Expression of GDF9 was highly correlated with BMP15, BMPR1B, and BMPR2 in large antral follicles. Expression of BMP7 in small antral follicles and BMPR2 in ovary tissues was significantly increased in the high-AFC group. Expression of ESR1 and ESR2 involved in estrogen production as well as PRKAA1 which involved in AMPK production were significantly greater in large antral follicles of high-AFC. There was a very high correlation between Cyp19 and ESR1 in large antral follicles and ovary tissues. Expression of Cyp19 and PRKAA1 were positively correlated with GDF9, BMP15, and BMP7 in large follicles. In conclusion, this study suggests that apart from the BMP signaling, genes involved in AMPK and estrogen production can be pivotal players in ewe's follicular development process. In addition, a strong cross-talk can exist among AMPK, BMP signaling, and estrogen synthesis systems in ewe ovary.

The present study was performed in indicus-taurus heifers 1) to determine if the antral follicle count (AFC) exhibits repeatability from puberty to yearling age and 2) to evaluate whether the phenotypic and genotypic parameters used in genetic improvement programs are correlated with AFC. In study I, Braford heifers (3/8 Nelore x 5/8 Hereford, n = 137) were serially examined by ultrasonography (with 60-day intervals) from weaning (9 ± 1 mo of age) to yearling ages (20 ± 1 mo of age) to monitor the numbers of antral follicles. In study II, the AFC of animals from experiment I and contemporary (same farm, considered at same age and kept under same conditions heifers n = 270 18-24 months) was correlated with the records of a genetic selection program using four statistical models with different covariates: i) model 1 considered effects of contemporary group and covariates age, weight gain from birth to weaning and visual scores for carcass traits at weaning, ii) model 2 covered contemporary group, age, weight gain from weaning to yearling and visual scores for carcass traits at yearling. The effects, variables and covariates of models 1 and 2 were combined to form model 3. Model 4 included the model 3 with addition of paternal effect. In study I, AFC varied from 3 to 64 follicles among females but was highly repeatable (0.89-0.92) within individuals in the same group. In study II, the four models tested showed low correlations with AFC: 0.072, 0.056, 0.082 and 0.172 for models 1, 2, 3 and 4, respectively. However, the model with paternal effect provided 17% of correlation of AFC and genotypic and phenotypic characteristics. Models 1, 3 and 4 also showed that AFC in indicus-taurus heifers can be influenced by finishing precocity at weaning (P

The aim of this study was to investigate the compatibility of the visual evaluation result of the blood flow characteristics and the blood flow measurements of the CL and the predictability of the responses given by corpora lutea with varying levels of blood flow to an induction of luteolysis by a PGF2α injection and to determine the possibility of increase in serum estrogen and progesterone concentrations in parallel with increased luteal blood flow (LBF). The cows, bearing a CL (n = 60; postpartum 35 days), were injected with PGF2α and were monitored for signs of estrous following the first injection. The cows, which did not show estrous signs, were examined for the presence of a CL on Day 14, whereas those that showed signs of estrous were examined on Day 10 following the onset of estrous. The level of LBF was visually graded as + (low; GI), ++ (medium; GII), +++ (high; GIII), and ++++ (very high; GIV). Immediately after the examination of LBFs, a second intramuscular injection of PGF2α was injected. In the cows, which were determined to be in estrous, the diameter of the Graafian follicles was measured by B-mode ultrasonography. Subsequently, these animals were artificially inseminated. The animals, which did not show estrous after the second injection, were examined as previously described and monitored for signs of estrous. A strong correlation (r = 0.654; P < 0.001) was determined to exist between the results of the visual examination of the images and the results obtained for the LBF area with the use of the Pixel Flux software. GIII (0.83 ± 0.15 cm2) and GIV (1.03 ± 0.48 cm2) were found to differ from GI (0.47 ± 0.23 cm2) and GII (0.51 ± 0.12 cm2) for the size of the LBF (P < 0.001). Serum progesterone levels in groups (GI, GII, GIII, and GIV) were determined to be 4.44 ± 2.42 ng/mL, 6.03 ± 2.37 ng/mL, 7.01 ± 2.94 ng/mL, and 7.17 ± 1.69 ng/mL, respectively. The comparative evaluation of the study groups showed that the groups did not statistically differ for the period between PGF2α injection and the onset of estrous, mean Graafian follicle size and estrogen levels. No direct correlation existed between these reproductive parameters and LBF.

Effect of a GnRH-agonist (deslorelin) was studied on reproductive function and ovarian luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) expression in prepubertal female cats that were either implanted with 4.7-mg deslorelin (implanted: n = 6) or not (controls: n = 18) or ovariohysterectomized at prepubertal age (prepubertal OVH: n = 6). Body weights, fecal estradiol, and sexual behavior of implanted and control cats were monitored for 48 weeks followed by collection of ovaries and uteri. Ovaries and uteri were collected from control cats at follicular, luteal, and inactive stage (n = 6/group) and from prepubertal OVH cats at prepubertal age. Ovaries and uteri were analyzed for anatomical/histological characteristics. Ovaries were also analyzed for LHR and FSHR expression. Statistical analysis showed higher (P ≤ 0.05) body weight in control than implanted cats only during 22nd to 26th weeks of the study. Estrus was observed in control cats only. Deslorelin reduced (P ≤ 0.05) ovarian weight and number of antral follicles but did not affect endometrial thickness and gland diameter. However, myometrial thickness of implanted cats was significantly lower than control cats at follicular and luteal stage. Ovarian LHR mRNA expression was lower (P ≤ 0.05) in implanted cats than control cats at follicular stage. FSHR mRNA and LHR protein expression did not differ among the three groups. FSHR protein expression was lower (P ≤ 0.05) in prepubertal OVH cats and was not affected by deslorelin. In conclusion, deslorelin suppresses reproductive function in prepubertal female cats for at least 48 weeks possibly through a change in the ovarian mRNA expression of LHR.

The oocytes from small antral follicle have low developmental potential to reach blastocyst due to incomplete cytoplasmic maturation during in vitro maturation (IVM). Thus, we developed an in vitro culture system for porcine oocytes derived from small antral follicles with l -ascorbic acid supplement during pre-maturation (pre-IVM) to support their development to blastocyst stage. Besides that, how l -ascorbic acid effect on the developmental competence of porcine oocytes with a special focus on histone modifications will be elucidated. The in vitro culture process consisted of two steps. The first step is 22 h of pre-IVM and the second step is 42 h of IVM. We utilized dibutyryl-cyclicAMP (dbcAMP) with L-ascorbic supplement during pre-IVM. Based on the result of this procedure, we proposed that the best culture condition in which hormone human chorionic gonadotropin (hCG) be added during the last 7 h of pre-IVM and continued culture to complete IVM. We observed that, in this culture system, the meiotic competence of porcine oocytes derived from small follicles was as high as those derived from large follicles after undergoing IVM. In addition, our study suggested that l -ascorbic acid supplementation at 100 μg/mL sharply enhanced the developmental potential of porcine oocytes. Interestingly, oocytes from small antral follicles treated with l -ascorbic acid could obtain the blastocyst quantity and quality as high as that of large antral follicles. The treated groups showed a significantly higher number of blastomeres compared to those in non-treated groups in both small and large follicle groups. Besides that, = The increasing levels of acetylation of histone H3 at lysine 9 (H3K9) and methylation of histone H3 at lysine 4 (H3K4) in blastocyst derived from small and large antral follicle under the present of l -ascrobic acid lead to a significant positive effect on the developmental competence and improvement in quality of porcine embryos.

Numerous studies have reported the importance of thyroid hormones on the development of later preantral and antral follicles, but their interactions with other hormones and effects in regulating early preantral follicle growth remain unclear. Here we investigated the in vitro effects of thyroxine combined with insulin on caprine preantral follicle survival and development. Sliced ovarian tissues were cultured for 1 or 7 days using 10 ng/mL (low) or 10 μg/mL (high) insulin in the presence of thyroxine at 0, 0.5, 1 or 2 μg/mL. Post-culture, we evaluated the follicular survival and development, assessed the expression of apoptotic-related genes (Bcl2/Bax) and receptors of insulin and thyroid hormones, and quantified the estradiol and reactive oxygen species (ROS) production levels. Follicular survival in low-insulin culture conditions was enhanced by the presence of 0.5 μg/mL thyroxine (P 0.05) as compared to the thyroxine-free medium but remained similar to non-cultured control in the presence of 2 μg/mL (P 0.05). Significantly higher ROS production was measured from Day 1 to Day 7 in low-insulin culture media containing 0.5 or 2 μg/mL thyroxine (P 0.05). When compared to high insulin level, the presence of thyroxine in low insulin culture conditions yielded higher stromal cell density (P 0.05), increased estradiol production on Day 1, and higher Bcl2/Bax ratio on Day 7. Cultures with high levels of both insulin and thyroxine led to follicles and oocytes with larger diameters (P 0.05). The RNA transcript levels of insulin and thyroid receptors were reduced in the presence of high insulin cultures when compared to controls (non-cultured). In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.

Domestic yaks are the most important livestock species on the Qinghai-Tibetan Plateau. Adult female yaks normally breed in warm season and enter anestrous in cold season. Currently, how the ovarian activity is regulated at the molecular level remains to be determined. This study was conducted to investigate follicular development and gene expression patterns of yak ovarian tissues in the warm and cold seasons. Dynamics of follicular development was evaluated based on histological analyses and global gene expression was examined by using RNA-sequencing (RNA-seq) technology. Firstly, we found that follicle development of yak cows in cold season was different from that in warm season. Interestingly, ovaries collected from yaks in cold season contained a significant higher number of antral follicles and some of these follicles showed signs of polycystic structure, indicating abnormal granulosa cell function. RNA-seq analyses of ovarian tissues from non-pregnant adult yaks in cold and warm season revealed that a list of 320 transcripts were differentially expressed, specifically, 79 were up-regulated and 241 were down-regulated in the ovaries from yaks during the cold season. Further analysis demonstrated that transcripts associated with estrogen secretion and metabolism signaling pathway were altered, including FST, CYP1A1, PIK3R1 and PIK3R2. This study showed histological features of follicle development and revealed candidate genes that may have important roles in regulating ovarian activities in the yak seasonal reproduction.

This study evaluated the effect of leptin on the in vitro culture of isolated sheep early antral follicles. Early antral follicles (300–450 μm) were isolated and cultured for 12 days in tissue culture medium 199 (TCM 199) supplemented with glutamine, hypoxanthine, transferrin, insulin, selenium, ascorbic acid, bovine serum albumin (BSA) and recombinant follicle stimulating hormone (rFSH) (TCM 199+: control medium) or TCM 199+ supplemented with 2 or 10 ng/mL leptin. After culture, oocytes were subjected to in vitro maturation (IVM). The parameters analyzed were morphology, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥110 μm) rates. After IVM, reactive oxygen species (ROS) levels, mitochondrial activity, meiotic stages and meiotic resumption rates were also analyzed. After 12 days of culture, the concentration of 2 ng/mL of leptin showed a higher percentage of morphologically normal follicles, fully-grown oocytes (≥110 μm), active mitochondria and meiotic resumption compared to the control medium (TCM 199+; P 0.05). After culturing, no significant differences existed among treatments in terms of the follicle diameter and ROS levels. In conclusion, the addition of 2 ng/mL leptin to the base culture medium is capable of improving follicular survival, oocyte growth, mitochondrial activity and meiotic resumption after the in vitro culture of isolated sheep early antral follicles.

This study set out to investigate the efficiency of long-term estrus synchronization protocols and ovulatory follicle dynamics in ultrasonographically monitored Santa Ines ewes during lengthening (LD; September-October) and shortening photoperiods (SD; April-May), and the transitional period (TP; January). In addition, the influence of ovarian status (e.g., size of antral follicles and/or presence of corpora lutea) at the outset of the estrus synchronization protocols on the ensuing development of ovulatory follicles was examined. Seventy sexually mature Santa Ines ewes were subjected to one of the two estrus synchronization regimens; on Day 0 (random day of the estrous cycle or anovulatory period), the ewes were fitted with an intravaginal progesterone (P4)-releasing (controlled intrauterine drug release [CIDR]) device, which was left in place for 14 days (G-1CIDR, n = 35) or replaced on Day 7 (G-2CIDR, n = 35), and received an intramuscular injection of 10 mg of PGF2α. The ewes allocated to the G-1CIDR group had mean serum P4 concentrations less than 2 ng/mL during the last 4 days of the synchronization protocol. There were no differences (P > 0.05) in mean ovulation rates between the two protocols tested nor among the ewes varying in ovarian status or studied at different times of the year, but ovulations occurred ∼ 12 hours later in the TP compared with the SD period (P < 0.05). Ovulatory follicles emerged earlier (P < 0.05) in the G-1CIDR group than in the G-2CIDR group (Day 8.3 ± 0.5 vs. 9.2 ± 0.4) and during LD (Day 7.1 ± 0.6) compared with the TP (Day 9.1 ± 0.6) and SD (Day 9.9 ± 0.5 of the protocol). In conclusion, the replacement of CIDR devices prevented the occurrence of lower-than-normal luteal phase levels of P4 at the end of the 14-day estrus synchronization protocol. However, although this procedure and seasonal influences altered certain growth characteristics of ovulatory follicles, there were no effects of these factors on the mean ovulation rate.

Quantitative patterns of expression of the growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) genes in different development stages of in vivo and in vitro grown ovarian follicles in sheep were studied for the first time. Both GDF9 and BMP15 were expressed in the cumulus cells and oocytes at all the development stages of in vivo and in vitro grown ovarian follicles. Growth differentiation factor 9 and bone morphogenic protein 15 exhibited stage-specific undulations in the expression in the cumulus cells and oocytes isolated from in vivo grown ovarian follicles. These undulations could be related to discrete development events during the ovarian follicle development. The expression of GDF9 and BMP15 was highest (3.38 ± 0.02 and 2.69 ± 0.06, respectively; P ≤ 0.05) in the primordial follicles compared with preantral, early antral, antral, and large antral stages. Similarly, GDF9 and BMP15 expression in the cumulus cells (0 ± 0.16 and 0 ± 0.07) and oocytes (1.47 ± 0.07 and 1.32 ± 0.03) was lowest (P ≤ 0.05) in the in vivo grown antral follicles. In the cultured follicles, the stage-specific undulations observed in the expression of GDF9 and BMP15 in the in vivo grown follicles were either different or abolished. For example, in the oocytes from in vitro grown follicles, the expression of BMP15 did not change as the development progressed all the way from preantral to large antral follicle stage although in the oocytes from in vivo grown follicles BMP15 expression exhibited stage-specific variations. It is concluded that GDF9 and BMP15 follow a stage-specific pattern of expression during the in vivo development of ovarian follicles in sheep, and in vitro culture altered the stage-specific changes in the expression of these two genes.

C1q/tumor necrosis factor-related protein 6 (CTRP6) is a newly identified adiponectin paralog with modulating effects on metabolism and inflammation. CTRP6 transcript is detected in human ovarian tissue. However, the expression pattern and function of CTRP6 on ovary have been rarely studied. In the present study, we preliminarily examined the structure feature and function of CTRP6 in porcine granulosa cells. The results indicated that the signaling peptide of CTRP6 was located at among positions 21 and 22, and the phosphorylation sites were at 15 (Ser), 4 (Thr) and 4 (Tyr), respectively. Meanwhile, CTRP6 was extremely homologous in livestock and chiropteran. The qPCR results showed that CTRP6 was moderately expressed in porcine follicle. Immunohistochemistry manifested that CTRP6 was presented in various types of ovarian cells. Immunofluorescence revealed that CTRP6 was located in cytoplasm in primary porcine granulosa cells. ELISA results showed that the concentration of CTRP6 in the follicular fluid was gradually decreased with the growth of antral follicle. In addition FSH increased CTRP6 expression levels in a time- and dose-dependent manner in primary porcine granulosa cells, while LH had no effect on CTRP6 basal gene expression, which suggesting CTRP6 is an FSH-responsive gene in porcine granulosa cells. Our findings imply that the CTRP6 may be a candidate gene to regulate folliculogenesis and reproductive performance.

Equine chorionic gonadotropin (eCG) has been commonly used to induce estrus in several felid species. However, the mechanisms by which this gonadotropin regulates cat folliculogenesis are still unclear. We investigated the responsiveness of cat ovarian follicles at different developmental stages to various eCG concentrations supplemented in vitro. Follicles were mechanically isolated from the ovaries of 22 cats and categorized into three developmental stages based on their morphology and diameter: 1) two-layered secondary follicle (SF), 100-150 μm (n = 139); 2) multi-layered SF, 150-300 μm (n = 154); and 3) early antral follicle (AF), ≥300-500 μm (n = 123). The follicles were then encapsulated in 0.5% (w/v) sodium alginate and cultured for 12 days in Minimum Essential Medium supplemented with 0, 0.05, 0.1 or 0.5 IU/mL eCG. Follicle and oocyte diameters were assessed every 3 days. On Day 12, mRNA expression levels of FHSR, LHCGR, GDF9, BMP15, CYP17A1, CYP19A1 and STAR were analyzed using real-time PCR. After being cultured for 12 days, follicle growth and mRNA expression of two-layered SF were not influenced by eCG at all concentrations (P 0.05). However, the concentration of eCG at 0.05 IU/mL stimulated follicular growth and gene expressions in the multi-layered SF and early AF (P 0.05). Correspondingly, the diameter of oocytes in the multi-layered SF and early AF treated with 0.05 IU/mL eCG was unchanged. Considering the mRNA expression, the level of STAR was enhanced in the early AF (P 0.05) and tended to increase in the multi-layered SF (P = 0.08) cultured in 0.05 IU/mL eCG, whereas the expression of other genes was not affected. In sum, the responsiveness of cat follicles to eCG is apparent from the multi-layered SF stage onward. The eCG supplementation at 0.05 IU/mL appeared to be optimal for the follicle culture in the domestic cats.

This study analyzed IGF-1 protein immunostaining in sheep ovaries, the effect of IGF-1 alone or associated with FSH on the culture of secondary follicles, and the immunostaining of LHR protein in antral follicles before and after culture. Ovaries were collected for IGF-1 protein analysis. In experiment 1, secondary follicles were cultured in α-MEM+ (control) or α-MEM+ supplemented with IGF-1 (10, 50 or 100 ng/mL). In experiment 2, follicles were cultured in the same media of experiment 1 plus 750 ng/mL FSH. Moreover, LHR immunostaining was analyzed in fresh antral follicles and after culture in 50 ng/mL IGF-1 + FSH. The IGF-1 protein was immunolocalized in oocytes from all stages of follicle development and in the granulosa cells from secondary and antral follicles. IGF-1 did not influence (P > 0.05) follicular viability and growth (experiment 1). However, in experiment 2, 50 ng/mL IGF-1 + FSH stimulated oocyte growth (P 0.05). In conclusion, the IGF-1 protein is present in all ovarian follicle stages in sheep. Moreover, the association between 50 ng/mL IGF-1 and FSH has a synergistic effect in vitro, increasing the percentage of fully grown oocytes and the intensity of immunostaining of LHR protein in oocytes and granulosa cells of cultured antral follicles.

High-producing dairy cows frequently suffer metabolic alterations that cause different diseases, which could decrease the reproductive efficiency of the herd. Among these reproductive disorders, cystic ovarian disease (COD) has been related to alterations in metabolites and hormonal factors such as insulin, adiponectin and leptin. The aim of this study was to determine the protein expression of adiponectin and some of its downstream targets in ovarian follicles of control cows and cows with clinical diagnosis of COD. We also analyzed some key metabolic sensors in plasma and follicular fluid from both groups. In follicular cysts, we detected higher protein expression of adiponectin receptor 2 (AdipoR2), 5′ adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 (CPT1) and acyl-coenzyme A oxidase 1 (ACOX1) relative to control antral follicles (p < 0.05). This was related to higher plasma adiponectin concentration in cows with COD than in control cows (p < 0.05). On the other hand, insulin concentrations showed an opposite pattern (p < 0.05). Furthermore, we found alterations in local and systemic concentrations of several metabolites. In this regard, in follicular fluid of cystic cows, the concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher (p < 0.05), whereas the concentrations of glucose and triacylglycerol were lower than in follicular fluid from control cows (p < 0.05). Besides, in both follicular fluid and plasma of cows with COD, the concentration of cholesterol was higher than in control animals (p < 0.05). These results evidence a local altered scenario of some metabolic sensors in cystic follicles, which could generate an adverse microenvironment for the resumption of ovarian activity, possibly causing the persistence of follicles and the recurrence of COD. Fil: Gareis, Natalia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Angeli, Emmanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Huber, Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Salvetti, Natalia Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Rodríguez, Fernanda Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Hein, Gustavo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Rey, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina