Davide Rondina

Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries.

The preantral follicles are the major source of oocytes and its utilization has been investigated as an important tool to store large numbers of female gametes for further utilization in reproductive programs. The aim of the present study was to perform quantitative and qualitative analyses of the efficacy of a mechanical method for isolating of preantral follicles from the ovaries of fetuses and from nonpregnant and pregnant ewes, using as reference the population of preantral follicles in situ. In the isolation method the ovaries were cut into fragments in the tissue chopper. Then, the suspension was filtered through nylon mesh filters. The number of isolated follicles per ovary was 1655, 4735 and 4770, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The number of in situ preantral follicles per ovary was 32961, 16627 and 17794, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The follicle recovery rate (number of isolated preantral follicles/number of in situ preantral follicles x 100) was higher in adult ewes (26 and 28%, respectively, for nonpregnant and pregnant ewes) than in fetuses (5%). Histological analysis showed that very few preantral follicles (less than 0.26% in situ and 0.46% after the isolation procedure) were degenerated. In conclusion, this study showed that a mechanical method could be used effectively to isolate a large number of intact ovine preantral follicles. In the future, with improvements in culture systems, the isolation of a great number of oocytes enclosed in preantral follicles will make a valuable contribution to the rare breeds and endangered species, agricultural efficiency and basic research in folliculogenesis.

Isolated ovine primordial follicles cryopreserved in different concentrations of ethylene glycol.

Cryopreservation of primordial follicles represents an opportunity to preserve female gametes, and consequently to protect the reproductive capacity of humans and animals, as well as to safeguard genetic material from endangered animal species or rare breeds. The aim of this work was to assess the toxicity of different concentrations of ethylene glycol (EG) to primordial follicles, and verify the viability of these follicles after the freezing-thawing procedure. Primordial follicles were isolated from ovine ovaries and exposed to different EG concentrations to evaluate the cryoprotectant (CPA) toxicity before and after cryopreservation. After isolation of primordial follicle (control), the number (mean+/-S.E.M.) of viable primordial follicles/ml was 3764+/-795.21. The number of viable follicles in the toxicity test using EG at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 1096+/-447.9, 960+/-446.67, 948+/-366.14, 832+/-313.59, 856+/-280.67, and 700+/-255.02, respectively. The number of viable follicles at concentrations of 2.5 M was less than for controls. After cryopreservation, the numbers decreased to 0+/-0, 148+/-85.46, 764+/-246.69, 824+/-291.9, 844+/-296.27, and 588+/-200.65, respectively for 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M EG. The number of viable follicles at 0, 0.5, and 2.5 M was less than for controls. In conclusion, after the freezing and thawing procedure, concentrations of 1.0, 1.5, and 2.0 M EG can be successfully used for the cryopreservation of isolated follicles in sheep.

Cryopreservation of ovine primordial follicles using dimethyl sulfoxide.

OBJECTIVE To verify the viability of isolated primordial follicles after exposure to different concentrations of dimethyl sulfoxide (DMSO) and after cryopreservation. DESIGN Randomized control trial. SETTING Laboratorio Renzo Giuliani, University of Florence, Italy. ANIMAL(S) Thirty- to 40-day-old lambs. MAIN OUTCOME MEASURE(S) Isolated primordial follicles were stained with trypan blue to evaluate the effect of different DMSO concentrations before and after the cryopreservation. Histological structure and follicular mortality were evaluated. RESULT(S) After the isolation procedure (control), a mean (+/-SE) of 800 +/- 203.86 live primordial follicles/mL were obtained. The number of live follicles in the toxicity test using the DMSO at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 782 +/- 193.96, 754 +/- 172.03, 778 +/- 191.58, 736 +/- 191.92, 476 +/- 122.9, and 316 +/- 83.52, respectively. The number of live follicles at 2.5 M was lower than that in the control procedure. After cryopreservation, the numbers decreased to 0 +/- 0, 232 +/- 44.20, 636 +/- 161.82, 628 +/- 181.28, 208 +/- 11.57, and 184 +/- 47.07, respectively at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M. The number of live follicles at 0, 0.5, 2.0, and 2.5 M were lower than that in the control procedure. CONCLUSION(S) After cryopreservation, only DMSO concentrations of 1.0 and 1.5 M showed a number of live follicles similar to that of the control procedure.

Embryo recovery rate in Santa Inês ewes subjected to successive superovulatory treatments with pFSH

The effect of two successive superovulatory treatments was assessed in 15 Santa Inewes synchronized with 60 mg medroxyprogesterone acetate sponges for 14 days and superovulated with 200 IU of porcine FSH (pFSH) in decreasing doses at 12 h intervals during days 12-14 of progestagen treatment. Estrus was observed from 12 h after sponge removal and the ewes in estrus were hand mated with Santa Inrams of proven fertility. All embryos were recovered by laparotomy 6-7 days after estrus. Treated ewes showed estrus from 24 to 72 h after sponge removal. The time of onset of estrus after sponge removal and the length of estrus were not affected ( P> 0.05) by the repetition of treatment. Ewes that failed to superovulate in the first and the second treatment ( P> 0.05) were 27 and 40%, respectively. The mean ovulation rate for those ewes responding to treatment was similar ( P> 0.05) between the first (9 .9 ± 1.1) and the second (11.3 ± 1.9) treatment. There was no significant difference in fertility between the first (53%) and second (40%) treatment. Among those fertilized, viable embryos were detected for 78% in the first treatment and 64% in the second treatment ( P> 0.05). It was concluded that Santa Inewes respond to superovulatory regimes with pFSH and the treatment may be repeated for at least two times.

Reproductive responses and progesterone levels of postpartum oestrus synchronization in goats with different body reserves

Thirty adult goats were classified at parturition into two body condition score (BCS) groups: BCI (n=16) with a score of 2.7 and BCII (n=14) with a score of 2.0. On the fiftieth day postpartum, oestrus was synchronized by CIDR for 5 days. Upon CIDR removal (Day 0), they received 1 mL of PGF2α IM and mated for 72 hours. Kids were kept with does and weaned at 40 days of age. Blood samples were taken at 0, 1, 4, 8 and 21 days after CIDR removal for progesterone assay. The BCI group showed a greater weight loss compared to the BCII group, and BCS before synchronization was 1.9±0.08 and 1.6±0.07 for the BCI and BCII groups, respectively (P<0.05). The weaning weight of BCI kids was greater when compared to BCII (P<0.001). After CIDR removal, all females were marked and mated. Pregnancy rate was higher in BCI goats (87% vs 36%; P<0.05), as well as prolificacy (1.65 vs 1.25; P<0.05) and twinning rate (0.62 vs 0.25; P<0.05). Progesterone concentration was higher in pregnant does in BCI. A positive relationship was found between progesterone level at CIDR removal and BCS at parturition (0.57; P<0.01), also between progesterone level at 21 days after CIDR removal and BCS at parturition (0.47; P<0.05), or BCS before synchronization (0.51; P<0.05). We conclude that oestrus response to postpartum CIDR synchronization appeared to be slightly dependent on BCS. However, goats with low BCS at oestrus synchronization exhibited a reduction in pregnancy rate.

Embryo production in superovulated goats treated with insulin before or after mating or by continuous propylene glycol supplementation.

Seventeen adult and cyclic Moxoto goats were synchronized using 60 mg MPA vaginal sponge for 11 days and 50 mug cloprostenol, 48 h before sponge removal, and superovulated with 120 mg pFSH i.m. in decreasing doses at 12 h intervals for three consecutive days. In seven goats, 0.2 IU/kg BW/day of long acting insulin was subcutaneously injected at same time as pFSH, and in the other five goats, the same dose of insulin was injected for three consecutive days starting 24 h after mating. Finally, five goats were supplemented with an oral dose of 80 ml/goat/day of propylene glycol continuously during the experiment. The animals were flushed at 7 days after mating and the embryos were classified based on International Embryo Transfer Society criteria. Blood samples were collected every 3 days for insulin assay. Administration of insulin raised the insulin levels of the goats (p < 0.05), whereas in the group treated with propylene glycol, insulin rate was different only between FSH treatment and after mating (p < 0.05). Similar rates of recovery for total (80.05 +/- 9.78%) or transferable structures (61.03 +/- 15.13%) were obtained. Treatment was not influenced (p > 0.05) by responsiveness to superovulation, which averaged 64%. By contrast, insulin treatments were shown to increase the number of embryos considered excellent with respect to goats supplemented with propylene glycol (p < 0.05). When insulin was given before mating, a strong relationship (r = 0. 90) (p < 0.05) between number of transferable embryo and ovulations was observed in the animals. In conclusion, superovulated goats treated with low doses of exogenous insulin resulted in an enhancement in embryo quality, which was related to changes in circulating insulin concentrations.

Sincronização do estro, indução da ovulação e fertilidade de ovelhas deslanadas após tratamento hormonal com gonadotrofina coriônica eqüina

The aim of this experiment was to evaluate the effect of different doses of eCG on reproductive performance of ewes. The estrus was synchronized with vaginal sponges with 30mg FGA during 12 days. At the time of sponge removal, ewes were distributed in three groups: 0 (n=26), 200IU eCG (n=30) and 400IU eCG (n=30). The estrus was detected by aid of a vasectomized ram. The ewes were inseminated by laparoscopy, 60h after sponge removal. Blood samples were collected at 5 and 18 days after insemination, in order to determine progesterone concentration. It was observed 70.9% of ewes in estrus after the end of treatment. The females that received eCG showed higher (P<0.05) percentage of estrus: 96.7% (400IU) and 76.7% (200IU) versus 34.6% (0 IU). The interval between the end of treatment and estrus onset was higher (P<0.05) in the group that did not receive eCG (54.7±6.3h) in comparison to the groups that received 200IU (45.9±7.8 h) and 400 IU eCG (40.4±10.3h). A lower (P<0.05) number of ovulating and pregnant ewes in the group that did not receive eCG was observed. The eCG withdraw resulted in negative effect on reproductive performance of ewes.

Pseudopregnancy in Saanen goats (Capra hircus) raised in Northeast Brazil.

The prevalence of pseudopregnancy over 44 months was investigated in 23 Saanen goats raised in Northeast Brazil during continuous oestrous cycling (cyclic group) or after synchronization of oestrus (synchronized group). The goats were monitored by ultrasonography and their plasma progesterone profile. The overall prevalence of pseudopregnancy was 30.4% (7/23). In the cyclic group, 28.6% (4/14) of goats showed pseudopregnancy, while in the synchronized group the prevalence was 33.3% (3/9). There was no significant difference between the groups (p>0.05). The mean (±SD) length of pseudopregnancy, as shown by the progesterone profile, was 121.6±33.5 days, ranging from 70 to 155 days. The study defined the prevalence of pseudopregnancy in Saanen goats raised in Northeast Brazil for the first time. This finding identified a major problem for this breed, as without treatment such animals remain unproductive until the spontaneous resolution of the condition. More research seems desirable to ascertain the prevalence of this condition in other breeds in this region of Brazil.

Hormonal changes in female buffaloes under shading in tropical climate of Eastern Amazon, Brazil

The hormonal responses of 20 female buffaloes raised under the sun (SS group) or in the shade (CS group) in Belem, Para, Brazil, were assessed. The CS group (n = 10) was kept in a silvopasture system featuring Racosperma mangium, whereas the SS group (n = 10) was kept in paddocks without shade featuring Brachiaria humidicola, drinking water, and mineral salt. The following parameters were measured for each treatment: dry-bulb temperature (AT), relative humidity (RH), and black-globe temperature in order to calculate the globe temperature and humidity index (GTHI). Blood sample collections to quantitatively determine levels of cortisol, triiodothyronine (T3), and thyroxine (T4) were performed every 14 days, at 13.00 h. Different seasons of the year were also assessed: rainy (January-April), transition (May-July), and less rainy (August-December). Analysis of variance revealed that AT and the index of global temperature and humidity (GTHI) differed between groups; specifically, higher values were found in the SS group, which shows how important silvopastoral systems are in buffalo farming in tropical climates. Relative humidity was significantly different across seasons and particularly high in the rainy season. The treatments influenced cortisol such that higher values were observed in the SS group. The highest mean cortisol levels were recorded during the rainy and less rainy seasons, whereas the highest T3 and T4 levels were recorded only during the rainy season. Triiodothyronine and thyroxine were negatively correlated with AT and GTHI and positively correlated with RH. Silvopastoral systems provide thermal comfort to buffaloes, and the less rainy season is more likely to cause heat stress.

Respostas reprodutivas e metabólicas de ovelhas alimentadas com bagaço de caju desidratado, durante o pós-parto

The effect of the addition of dehydrated cashew apple bagasse (DCB) to the diet on ewe postpartum response was evaluated on 41 ewes, housed with their kids in pens, where they received two diets containing 75% of Elephant Grass plus 25% of concentrate (Diet I; n=17), or 50% of DCB plus 25% of Elephant Grass plus 25% of concentrate (Diet II; n=24). On the 50th day postpartum, estrus was synchronized and ewes mated. Diet Igroup exhibited a greater loss of live weight (P 0.05) for the presence of first functionally CL, estrus synchronization response, and gestation or prolificity rates. Thus, the addition of DCB to the diet was well accepted, induced a reduction of weight loss, and did not affect the reproductive response of ewes.