[Show abstract][Hide abstract] ABSTRACT: Mules are hybrid females, commonly considered as infertile, resulting from a cross between a donkey and a mare. Few recent studies address the particularities of the reproductive system of the mules. Here, we present data showing consistent evidences of regular cyclicity in mules and also the first report of two births of donkey foals after embryo transfer into mule recipients with spontaneous estrous cycle. Ovaries of mules (n = 72) were obtained at a local slaughterhouse. Clear evidences of cyclicity were found in 61.1% (44/72) of the ovaries, being 45.8% (33/72) corpora lutea and 15.3 (11/72) large antral follicles. After follicle aspiration, we obtained oocytes with regular distribution of granulosa cells and homogenous characterization of ooplasm. Because of the evidence of cyclicity in mules, two embryos collected from a donkey were transferred into two mules with spontaneous estrous cycles. Eutocic births of donkey foals occurred after 372 and 379 days of gestation. In contrast to the classical concept of infertile females, we found clear evidences that a higher percentage of mules can present ovarian activity and these animals may be considered for reproductive biotechnologies, such as embryo recipients.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to compare the conception rates to fixed-time artificial insemination (FTAI) and in vitro embryo production between Nelore cows with high or low antral follicle counts (AFC). First, multiparous Nelore cows (Bos indicus, n=547, 40-60 days postpartum) were subjected to synchronization of ovulation. Randomly during their oestrous cycle (Day 0), cows received an intravaginal device containing 1.9g of P4 (CIDR®) and 2mg of oestradiol benzoate (Estrogin®), intramuscularly. At device removal (Day 8), cows received 500µg of PGF2α (Ciosin®), 300IU of eCG (Novormon®), and 1mg of oestradiol cipionate (ECP®), intramusculary. All cows were inseminated 48h after P4 device removal. Antral follicles=3mm were counted using an intravaginal microconvex transducer (Day 0), and cows were assigned to groups of high (G-High,=25 follicles, n=183), intermediate (G-Intermediate, 16-20 follicles, n=183), or low AFC (G-Low,=10 follicles, n=181). In another study to compared the in vitro embryo production, Nelore cows (n=66, 72-96 months) were subjected to ultrasound-guided follicular aspiration using an intravaginal microconvex array transducer (7.5MHz). The COC were selected and cows were assigned to groups according to the oocyte production: G-High (n=22,=40 oocytes), G-Intermediate (n=25, 18-25 oocytes), or G-Low (n=19,=7 oocytes). Previously tested semen from a single bull was used for IVF using a previously described protocol (Silva-Santos et al. 2014 Reprod. Domest. Anim. 49, 228-232). The oocyte and embryo production (viable embryo: grade I, II, III; vitrifiable embryo: grade I, II) were evaluated. The number of follicles was evaluated by Kruskal-Wallis, and the chi-square test was used for data on oocyte and embryo production (P=0.05). The average follicular population was 30.7±5.7 (G-High), 18.6±1.64 (G-Intermediate), and 7.8±2.4 follicles (G-Low; P<0.05), but there were no differences in the conception rates among groups (51.9 v. 48.6 v. 58.6%, respectively; P>0.05). The total number of oocytes recovered were 1109 (G-High), 534 (G-Intermediate), and 101 (G-Low; P<0.05). The mean number of viable oocytes was 40.4±10.6 (G-High), 14.8±3.0 (G-Intermediate), and 3.8±1.1 (G-Low; P<0.05) and the percentage of viable oocytes was 80% (G-High), 69% (371/534, G-Intermediate), and 71% (G-Low; P<0.05). Cleavage rate was 79% (G-High), 74% (348/472, G-Intermediate), and 71% (G-Low; P<0.05), and blastocyst rate was 42% (G-High), 32% (153/472, G-Intermediate), and 13% (G-Low; P<0.05). The number of viable embryos was 18.4±6.7 (G-High), 6.1±3.6 (G-Intermediate), and 0.6±0.7 (G-Low; P<0.05) and the percentage of vitrifiable embryos was 81% (G-High), 77% (118/153, G-Intermediate), and 58% (G-Low; P<0.05). Therefore, Nelore cows with high oocyte production had ~10-fold higher oocyte production and produced ~30-fold more embryos compared with the low AFC group. In conclusion, AFC had no influence on the conception rates to FTAI; however, Nelore cows with high oocyte production exhibited higher in vitro embryo production.
Reproduction Fertility and Development 12/2014; 27(1):206. DOI:10.1071/RDv27n1Ab233 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multioocyte follicles (MOF) are follicles that enclose two or more oocytes. They have been described in many mammalian species, but there is no evidence about their activity in the ovaries. The aim was to estimate the prevalence of MOF and to compare the cell proliferation activity between follicles containing one or more oocytes in the ovaries of prepubertal and adult bitches. Eighty ovaries from prepubertal (n=20) and adult bitches (n=20) were obtained by elective ovariohysterectomy (OHE). Immediately after OHE, ovaries were immersed in Bouin's fixative for histological processing. 5µm thick sections were mounted on histological slides and stained with periodic acid-Schiff (PAS) and hematoxylin. Cell proliferation was evaluated by immunohistochemistry using the proliferating cell nuclear antigen (PCNA). Monoclonal antibody PCNA (clone PC1O, 1:200 dilution, Biocare, Concord, CA, USA) was used according to manufacturer's instructions and an antibody diluent was used as a negative control. Slides were counterstained with hematoxylin and examined at 200× to 400× magnification under light microscope. Only cells showing PCNA signal exclusively in the nucleus were considered positive. The prevalence of MOF in the ovaries was compared using a Fisher's exact test (P<0.05). In all females, the prevalence of MOF was 55% (22/40). MOF containing two or three oocytes were more abundant; however, multioocyte follicles with up to 12 oocytes were observed. The prevalence of MOF at the primordial stage was higher for prepubertal bitches (47 v. 28%) but adult bitches exhibited a higher frequency of secondary MOF (49 v. 25%; P<0.05). There was no difference in the prevalence of MOF at primary stage between prepubertal and adult bitches (28 v. 23%; P>0.05). Regarding the cell proliferation activity, PCNA immunoreactivity was detected in oocyte nucleus and granulosa cells of multioocyte follicles at different stages of development. Similarly to what was observed for follicles containing only one oocyte, all nuclei of oocytes within multioocyte follicles exhibited PCNA immunoreactivity and there was a gradual increasing of immunoreactivity in granulosa cells according to the stage of follicular growth. Expression of PCNA by granulosa cells of multioocyte follicles was higher in the secondary and antral stage of development; however, some primordial and primary follicles also exhibited some PCNA-positive cells. In conclusion, the prevalence of MOF at the primordial stage of development was higher in prepubertal bitches, whereas MOF at the secondary stage were more frequent in adult bitches. The PCNA expression pattern by the oocyte nucleus of multioocyte follicles was similar to that observed in follicles containing only one oocyte, which is suggestive of similar activity between these follicles. Furthermore, the presence of proliferative activity in granulosa cells of multioocyte follicles suggests an association of the PCNA expression with more advanced stages of follicular growth.
Reproduction Fertility and Development 12/2014; 27(1):183-4. DOI:10.1071/RDv27n1Ab185 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objectives of this study were to compare populations of preantral follicles between purebred Bos indicus and Bos taurus cows with high or low antral follicle counts (AFC) and to correlate the number of preantral follicles with the population of antral follicles. Nelore (Bos indicus, n = 100) and Angus (Bos taurus, n = 100) cow ovaries were collected at abattoirs and examined using ultrasonography. Antral follicles ≥ 3 mm were counted, and the cows ovaries were assigned to high (G-High) or low (G-Low) AFC groups based on the mean number (± 1 SD) of ovarian antral follicles: Bos indicus with high AFC (≥ 57 follicles, n = 8) or low AFC (≤ 21 follicles, n = 8) and Bos taurus with high (≥ 45 follicles, n = 10) or low AFC (≤ 13 follicles, n = 10). The ovaries were processed, and the number of preantral follicles was estimated. Between-groups comparisons were performed using a Kruskal-Wallis test, and the correlation between preantral and antral follicles was evaluated using a Pearson's correlation test (P ≤ 0.05). A large variation in the number of preantral follicles was observed among the animals. Although there was a correlation between the population of preantral follicles and the number of antral follicles, there was no difference between the mean number of preantral follicles in the Bos indicus G-High (48,349 ± 30,149) and G-Low groups (33,037 ± 31,710) or between the Bos taurus G-High (35,050 ± 36,060) and G-Low groups (30,481 ± 43,360). Therefore, the preantral follicle population did not differ between purebred Bos indicus and Bos taurus cattle with high or low AFC but was correlated with the number of antral follicles.I In addition to the large within-groups variation in the number of preantral follicles, some cows with high AFC had lower populations of preantral follicles compared to the low AFC group, and the highest population of preantral follicles was observed in both Bos indicus and Bos taurus with low AFC.
[Show abstract][Hide abstract] ABSTRACT: Synchronization of estrus and ovulation are of paramount importance in modern livestock improvement programs. These methods are critical for assisted reproduction technologies, including artificial insemination and embryo transfer, that can increase productivity. In the current study, subcutaneous implants containing norgestomet were placed for long (14 days), medium (9 days), and short (5 days) periods of time in 70 crossbred ewes undergoing fixed-time artificial insemination. The resulting effects on estrus synchronization and conception rates were subsequently evaluated. Among the synchronized ewes, 85.7% (60/70) underwent estrus over a period of 72 h after progestagen treatment ceased. The shortest mean interval between withdrawal of the device and onset of estrus (34.2 ± 8.9 h) was observed in the G14 days of P4 group (p < 0.05). The conception rate of the G14 days of P4 group was statistically higher than that of the other groups (83.3% vs. 60.9% vs. 47.8%; p < 0.05). In conclusion, 14 days of norgestomet treatment produced higher conception rates and a greater number of pregnancies at the beginning of the breeding season.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to compare the lipid profile in oocytes of indicus and 1/2 indicus × taurus cows with high and low antral follicle count (AFC)/oocyte yields. After an OPU procedure (D0), antral follicles ≥3 mm were counted by ultrasonography (D4, 19, 34, 49, 64), and cows were assigned to groups with either high AFC (≥30 follicles; indicus, NH group; 1/2 indicus × taurus, AH group) or low AFC (≤15 antral follicles; indicus, NL group; 1/2 indicus × taurus, AL group). The lipid profiles of the oocytes were determined by MALDI-MS. For GI, GII and GIII oocytes, the indicus samples tend to cluster separately from the 1/2 indicus × taurus samples. The lipid species [PC (P-38:5) + H](+) and/or [PC (P-36:2) + Na](+) , [PC (38:2) + H](+) , [PC (38:5) + Na](+) and [TAG (60:8) + NH(4) ](+) were more abundant in indicus (NH and NL groups) than 1/2 indicus × taurus. The higher lipid content in the indicus oocytes likely reflects differences in the rate of lipid metabolism and may contribute to oocyte competence and embryo development.
[Show abstract][Hide abstract] ABSTRACT: Synchronization of estrus and ovulation is a reproductive technology of paramount importance in modern livestock improvement programs. It has a critical role for the implementation of assisted reproduction technologies that could increase productivity, including artificial insemination and embryo transfer. To this end, the aim of this study was to evaluate the efficacy of protocols using subcutaneous implants containing Norgestomet for long (14 days), medium (nine days) and short (five days) durations on the estrus synchronization and conception rates of 70 crossbred ewes undergoing fixed-time artificial insemination. Among the synchronized ewes, 85.7% (60/70) showed estrus over a period of observation lasting 72 h after the progestagen was removed. The shortest mean interval between withdrawal of the device and onset of estrus was observed in the G14 group at 34.2 ± 8.9 h (P < 0.05). The conception rate was statistically greater in the G14 group than in the other treatment groups (83.3% vs. 60.9% vs 47.8%; P < 0.05). In conclusion, the 14-day Norgestomet protocol demonstrated to be the best option, providing higher conception rates and consequently a greater number of pregnancies in the beginning of the breeding season.
[Show abstract][Hide abstract] ABSTRACT: In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 μM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to compare the number of follicles, oocytes and the recovery rate in sheep submitted to the one-shot protocol with or without ovarian priming with estradiol benzoate (EB). Pluriparous non-lactating sheep (n=33) with an average age of five years (range 4-6) and a body condition score of 3.0 +/- 0.3 were divided into three groups. The one-shot group (n=10) was treated with a subcutaneous implant containing 1.5 mg of norgestomet from D0 to D10. The animals in this group were administered 0.04 mg of D-cloprostenol, 200 IU of follicle stimulating hormone (FSH) and 300 IU of equine chorionic gonadotropin (eCG) on D8. Animals in the EB group (n=11) received the same treatment as one-shot plus the administration of 0.6 mg of EB on D0. In the untreated group (n=12), the animals received no hormone stimulation. The collection of the oocytes was performed by laparotomy 36 h after the administration of gonadotropins (D10). Oocytes were searched and classified based on morphology. An increase was observed (p<0.05) in the number of follicles aspirated in the one-shot vs. the EB and untreated groups (16.3 +/- 5.6 vs. 9.5 +/- 2.4 and 12.1 +/- 4.1), respectively. The average number of oocytes and the recovery rate were higher (p<0.05) in the one-shot and EB groups compared to the untreated group, resulting in 14.2 +/- 9.0 and 87.1% (142/163), 11.0 +/- 6.2 and 91.4% (122/134) vs. 6.8 +/- 3.5 and 71.9% (82/114), respectively. It was concluded that the EB did not improve efficiency in the one-shot protocol, but was significantly better than in untreated animals.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the feasibility of long distance transport of ovine oocytes and in vitro produced embryos. Santa Ines pluriparous sheep (n = 5) were synchronized with intravaginal devices containing 0.3 mg progesterone (CIDR®, Pfizer, Netherlands) for 10 days. Thirty-six hours prior to progesterone device removal and follicular aspiration, 0.04 mg d-cloprostenol sodium (Ciosin®, Coopers, Brasil), 200 IU FSH (Folltropin®, Bioniche, Canada) and 300 IU eCG (Novormon®, Syntex, Argentina) were administered intramuscularly. Oocyte retrieval was performed via mid-ventral laparotomy, under general anesthesia. The oocytes were evaluated and classified under a stereomicroscope and placed in cryovials with TCM 199, aerated with 5% CO2, covered with mineral oil, sealed, wrapped with parafilm and stored in a transport incubator at 38.5 °C. Oocytes were then transported via the road for 14 h to the laboratory, where the oocytes then completed more than 10 h of IVM. In vitro fertilization (IVF) was performed with frozen sperm from a single ram. On Day 3 the embryos were placed in cryovials containing SOFovine (In Vitro Brasil, Sao Paulo, Brazil), aerated with 5% CO2, covered with mineral oil, sealed, wrapped with parafilm and placed into the same portable incubator. The embryos were returned to the farm under the same transport conditions (km and h). Upon arrival at the farm, all embryos were evaluated and transferred into recipient ewes 4 days after the initial oocyte retrieval. Embryos were transferred in sets of 3 to 4, into the apex of the uterine horn, ipsilateral to the corpus luteum, by means of mid-ventral laparotomy, under general anesthesia. Pregnancy was diagnosed using transrectal ultrasonography 30 days after embryo transfer. On average 9.8 ± 5.7 oocytes (49 oocytes/5 donors) were obtained per donor, and 25 grade I morulae were transferred into 7 recipients. One pregnancy (14.3%) was confirmed resulting in a healthy lamb. These results indicate the feasibility of in vitro embryo production in sheep after maturation and embryonic development following transport over long distances.
Small Ruminant Research 06/2012; 105(s 1–3):286–289. DOI:10.1016/j.smallrumres.2012.03.004 · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the reproductive performance of ewes treated with eCG and an exogenous progestagen protocol during the spring season. Forty-eight mixed-breed wool and hair ewes (body condition score of 2.8 ± 0.5 and 41 ± 3 kg) were randomly assigned into two groups (n = 24/group), which received (G-Sync) or not (G-Control) an intravaginal device (Day 0) containing 60 mg of medroxyprogesterone acetate. On Day 7, ewes of the G-Sync were injected with 300 IU of eCG and 30 ug of d-Cloprostenol, im. On Day 9, the device was removed and 12 h later males were introduced into the G-Sync and G-Control groups in a proportion of 1:6. Estrus response observation and mating were performed during Days 10, 11 and 12 from 7 to 9 AM and 4 to 6 PM. After Day 12, males were separated from females for 10 days and later reintroduced into the flock for 45 days. Estrus rates for the G-Control and G-Sync groups during Days 10, 11 and 12 were 4 and 88% (P < 0.05), respectively. Pregnancy rates from initial mating on Days 10, 11 and 12 were 0 (G-Control) and 46% (G-Sync; P < 0.05). Total pregnancy rates for the whole mating season were 50 (G-Control) and 79% (G-Sync; P < 0.05). The exogenous progestagen protocol plus eCG used for estrus induction/synchronization improved the pregnancy rate of mixed-breed wool and hair ewes by about 29% points at the end of the breeding season. Thus, this procedure seems to be appropriate to be implemented as part of the reproductive management of some ovine farms during the non-breeding season.