Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 °C, 5% CO₂, and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH + 10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH + 5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14 ± 0.074 and 0.15 ± 0.076 vs. 0.46 ± 0.078 and 0.50 ± 0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.     

Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares

The dynamics of ovarian follicular development depend on a timely interaction of gonadotropins and gonadal feedback in the mare. The development and efficacy of genetically cloned recombinant equine gonadotropins (reFSH and reLH) increase follicular activity and induce ovulation, respectively, but an optimum embryo recovery regimen in superovulated mares has not been established. The objective of this study was to determine if treatment with reFSH followed by reLH would increase the embryo per ovulation ratio and the number of embryos recovered after superovulation in mares. Sixteen estrous cycling mares of light horse breeds (4-12 years) were randomly assigned to one of two groups: Group 1; reFSH (0.65mg)/PBS (n=8) and Group 2; reFSH (0.65mg)/reLH (1.5mg) (n=8). On the day of a 22-25mm follicle post-ovulation mares were injected IV twice daily with reFSH for 3 days (PGF(2α) given IM on the second day of treatment) and once per day thereafter until a follicle or cohort of follicles reached 29mm after which either PBS or reLH was added and both groups injected IV twice daily until the presence of a 32mm follicles, when reFSH was discontinued. Thereafter, mares were injected three times daily IV with only PBS or reLH until a majority of follicles reached 35-38mm when treatment was discontinued. Mares were given hCG IV (2500IU) to induce ovulation and bred. Embryo recovery was performed on day 8 day post-treatment ovulation. Daily jugular blood samples were collected from the time of first ovulation until 8 days post-treatment ovulation. Blood samples were analyzed for LH, FSH, estradiol, progesterone and inhibin by validated RIA. Duration of treatment to a ≥35mm follicle(s) and number of ovulatory size follicles were similar between reFSH/reLH and reFSH/PBS treated mares. The number of ovulations was greater (P<0.01) in the reFSH/reLH group, while the number of anovulatory follicles was less (P<0.05) compared to the reFSH/PBS group. Number of total embryos recovered were greater in reFSH/reLH mares than in the reFSH/PBS mares (P≤0.01). The embryo per ovulation ratio tended to be greater (P=0.07) in the reFSH/reLH mares. Circulating concentrations of estradiol, inhibin, LH and progesterone were not statistically different between groups. Plasma concentrations of FSH were less (P<0.01) in the reFSH/reLH treated mares on days 0, 1, 4, 6, 7 and 8 post-treatment ovulation. In summary, reFSH with the addition of reLH, which is critical for final follicular and oocyte maturation, was effective in increasing the number of ovulations and embryos recovered, as well as reduce the number of anovulatory follicles, making this a more viable option than treatment with reFSH alone. Further evaluation is needed to determine the dose and regimen of reFSH/reLH to significantly increase the embryo per ovulation ratio.     

The influence of ovulation number and ovarian dimensions on embryo production in superovulated cattle

Twenty-one cycling Angus heifers and five Holstein cows received a subcutaneous (SC) injection of 50 mg of progesterone (P) in oil for 14 consecutive days. On day 6 of (P) treatment, animals were injected intramuscularly (IM) with 6 mg of estradiol valerate, and on day 13, received an IM injection of 2,000 IU of Pregnant Mare Serum Gonadotropin. Three additional Angus heifers were used as non-hormone treated controls. Seventeen of 21 heifers and 4 of 5 cows (81%) exhibited estrus within 48 to 132 hr following P treatment. Two of the five animals in which estrus was not observed were palpated as pregnant and discarded from the study. Treatment animals showing estrus were randomly assigned either to Group I, animals bred by natural service, or Group II, animals artificially inseminated with two straws of frozen semen at 12-hr intervals for a total of four breedings. Twenty-one animals were slaughtered 2 to 6 days after the onset of estrus, and those animals in which estrus was not detected were slaughtered 10 days after the last P injection. Two of the 24 treated animals had no ovulations. A total of 397 ovulation points ($\text{397}\text{22}$) were counted for a mean ovulation rate of 18 ovulations per animal. One hundred and fifty-six ova were recovered ($\text{156}\text{397}$) for a collection rate of 39%. Group I animals had 44 of 66 (67%) of their ova fertilized while 23 of 71 (32%) of the ova in Group II were fertilized. Nineteen unfertilized eggs were collected from the three animals not observed in estrus. No differences in fertilization rates between the Group I and Group II animals were found. Mean ovarian width, length and weight in the treated animals was measured and found to be 3.5 ± 1.1 cm, 4.8 ± 1.4 cm, and 21.7 ± 21.2 gm, respectively. Ovarian width, length and weight were all positively correlated with the number of ovulations per ovary r=.74, r=.74, and r=.55, respectively. No significant correlation existed between ovarian width (r=.16), lenght (r=.21), or weight (r=.13) when compared to ova recovery rate. This result suggests that ovarian size or weight may not be the limiting factor involved in embryo recovery.     

Effect of dietary protein on embryo recovery rate and quality in superovulated heifers

For almost 3 decades, superovulation and embryo transfer have been used in cattle breeding to increase the number of offspring from genetically superior female animals. Several factors including nutrition affect the number of transferable embryos recovered. We compared the effects of two different dietary protein levels easily achieved in practical conditions on embryo number and quality in superovulated heifers. Finnish Ayrshire heifers (n = 37) were allocated to isoenergic diets containing either 14% (D14) or 18% (D18) crude protein (CP). Estruses were synchronized, and the heifers were subsequently superovulated and inseminated using a standard FSH-protocol. Embryos were collected 7 days after inseminations (71-72 days after the beginning of the treatment period) by uterine flushing. The number of corpora lutea, and the number and quality of embryos were determined. Protein feeding did not affect superovulatory response, the number of embryos or the number of transferable embryos recovered. Proportionally more poor-quality embryos were found in group D14 than in group D18 (20.2% versus 13.2%, respectively, P = 0.053). It is concluded that a long-term moderate increase in the content of crude protein fed to energy-adequate heifers does not seem to affect superovulatory response and the number of embryos recovered, but it may be advantageous to the quality of embryos.     

Effects of inbreeding on ovarian responses and embryo production from superovulated Mantiqueira breed cows

The objective of the present experiment was to evaluate the effects of different levels of inbreeding on ovarian response and embryo production from superovulated cows. One hundred and thirteen Mantiqueira cows (a medium-size, Bos taurus native dairy cattle breed), with inbreeding coefficients ranging from 0 to 30%, were allocated into five classes of inbreeding and subjected to superovulation treatment. At induced estrus, cows were mated with Mantiqueira bulls (with minimal inbreeding). Six to eight days after mating, the cows were slaughtered, ovarian structures counted and embryos recovered by flushing the uterine horns and oviducts. Sire, season, age, weight, parity and age at first calving of donors did not significantly affect ovarian response or embryo production and quality. There were no effects of inbreeding class on number of total corpora lutea (CL) or number of CL present in the right ovary. However, the number of CL in the left ovary was reduced (P<0.05) in cows with Class 5 (>9%) of inbreeding. The number of transferable, but not the number of non-transferable embryos or the total number of embryos from cows with Class 5 of inbreeding, was lower (P<0.05) than those of cows from Classes 0 to 4 (<9%) of inbreeding. There was a quadratic decrease in the number of transferable embryos as inbreeding coefficient increased (Y=11.077+0.34X-0.0529X(2); R(2)=0.91, P<0.01), but no significant linear or quadratic effect of inbreeding on total number of embryos or number of non-transferable embryos. In conclusion, an inbreeding coefficient>9% reduced the quality of bovine embryos at the initial stage of development.     

Dose of FSH-P as a source of variation in embryo production from superovulated cows

Various superovulation treatments were evaluated retrospectively in a commercial embryo transfer program. When it appeared that embryo production was dependent on the dose of FSH-P, a dose response curve to FSH-P was developed and embryo production compared using several treatment regimes. There was a significant effect of dose of FSH-P on embryo production in superovulated cows. At doses in excess of 28 mg, embryo production declined from 5.9 transferable embryos per collection (28 mg) to 2.7 (60 mg). Total embryos collected declined from 14.9 to 6.8 and the percent transferable from 57% to 40%. There was no advantage in using a five-day treatment over a four-day treatment regimen or in using a level over a declining dose regimen. There was a large individual variation in cow response rendering decisions on treatment changes based on single records unreliable. The percentage of zero collections increased with dose rate. Adoption of a 28-mg dose rate in commercial donors resulted in the embryo production forecast by these studies.     

Stimulation of testosterone production by isolated rabbit ovarian follicles in the presence of luteinizing hormone and phenylmethylsulfonyl fluoride.

Luteinizing hormone (LH) causes a dramatic increase in steroidogenesis by isolated rabbit follicles which secrete testosterone as a major product. In order to determine whether the source of this testosterone could be from stores of cholesterol esters rabbit follicles were incubated with LH and phenylmethylsulfonyl fluoride (PMSF) an inhibitor of cholesterol esterase. No inhibition of testosterone production could be detected in the presence of PMSF indicating that cholesterol esters are not precursors for testosterone synthesis by rabbit follicles.     

Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers

We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n = 20) originating from a common herd were assigned to three treatment groups using a 3 × 3 latin square design replicated in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received a commercially available organic mineral supplement (organic; n = 52); or (3) heifers received an all inorganic mineral supplement (inorganic; n = 55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.5) and control (1.6 ± 0.5) treated heifers than organic (0.4 ± 0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements.     

Effect of phenoxybenzamine on luteinizing hormone release in the female rat

To clarify the mode of action of phenoxybenzamine, an alpha adrenergic blocking agent, its effects upon plasma LH levels in ovariectomized rats and upon the ovulatory LH surge expected between 1400 and 1600, the critical period, on the day of proestrus in normal rats were studied. A single injection of phenoxybenzamine, 20 mg/kg, given at 1300 on the day of proestrus bokced ovulation (1 out of 7 ovulating), while plasma LH did not differ from controls between 1500 and 1600. An additional injection of 20 iu HCG at 1500 prevented the ovulation block (83% ovulating). A single phenoxybenzamine injection at 1700 failed to prevent ovulation (5 out of 7 ovulating). The beta adrenergic blocking agents, propanolol and MJ 1999, did not affect ovulation. Treatment with phenoxybenzamine for 2 days, 20mg/kg/day, for 8 days, 10mg/kg/day, were did not prevent the rise causing a reduction in blood flow through the ovary rather than acting as a neurogenic stimulus in the hypothalamus.     

Ovarian response,embryo production and hormonal profile in superovulated goats treated with insulin

The influence of insulin on ovarian response and embryo production was investigated in 30 mixed breed goats, divided randomly into three equal (n=10) groups. Goats in Group 1 (control) were superovulated using 20 IU FSH i.m. in six divided descending doses, i.e. 4/4, 3/3 and 3/3 IU at 12 h interval for three consecutive days and were not given insulin treatment. Goats in Group 2 (insulin pretreatment) were pretreated with long acting purified bovine insulin 0.2 IU/kg body weight per day s.c. on Days 7, 8 and 9 of the estrous cycle prior to initiation of superovulatory treatment as in Group 1. Animals in Group 3 (insulin cotreatment) were treated as in Group I, but in addition received long acting purified bovine insulin 0.2 IU/kg body weight per day s.c. as a cotreatment along with the first, third and fifth FSH treatments on three consecutive days. Total ovarian response (corpus luteum and unovulated large follicle (UOLF)) was significantly (P<0.05) higher in insulin pretreatment (17.90+/-3.08) than in the cotreatment (11.50+/-2.34) and control (11.90+/-1.87) groups. The number of UOLF was significantly higher (P<0.05) in the insulin pretreatment (10.2+/-1.67) than the cotreatment (4.9+/-1.14) and control (3.6+/-1.09) groups. The mean transferable quality of embryos did not differ significantly among treatments. Progesterone concentration on the day of PGF(2)alpha treatment was not different (P>0.05) between the insulin treatment groups (5.28+/-0.79; 5.30+/-0.66 ng/ml). Estradiol-17beta concentration was significantly (P<0.05) higher on the day of PGF(2)alpha treatment in both the insulin treatment groups (36.67+/-6.40; 34.33+/-4.33 pg/ml) as compared to the control group (20.00+/-2.73 pg/ml). There is ample evidence to indicate beneficial effect of insulin on folliculogenesis and steroidogenesis in superovulated goats.     

Direct hypophysial inhibition of luteinizing hormone release by dopamine in the rabbit.

The role and site of action of dopamine in regulating gonadotropin secretion remain unclear. In the present study, we investigated the possibility that dopamine regulates LH secretion by acting directly on the pituitary gland of the rabbit. The effect of dopamine infusion on LHRH-evoked LH release was determined in intact and pituitary stalk sectioned animals. Intravenous injection of LHRH (1 μg) in intact and acutely stalk sectioned rabbits increased peripheral plasma LH levels from a resting value of 0.2 ng/ml to maximal values of 12–14 ng/ml within 10–20 min. When dopamine was infused iv at a dose of 6.6 μg/min/kg BW from 30 min before LHRH injection until 120 min after, the rise in plasma LH levels in intact and stalk sectioned animals was decreased by 50–70%. However, dopamine infused at a lower dose (0.66 μg/min/kg BW) or at a higher dose (66.0 μg/min/kg BW), did not affect the LHRH-induced secretion of LH. These results suggest that dopamine can exert a direct hypophysial inhibitory effect on release of LH. They also demonstrate that dopamine is inhibitory only within a restricted dose-range, extending to the pituitary an established property of dopamine in the cardiovascular system.     

The effect of prostaglandin F2 alpha treatments in superovulated cattle on estrus response and embryo production

Approximately 10% of cows in a commercial embryo transfer center that were superovulated for embryo production did not show estrus at the right time and therefore did not produce embryos. This problem was investigated by studying the effects of prostaglandin F2 alpha (PGF) treatment regime and dose rate on the superovulatory process. The cows in estrus following superovulation (96% vs. 86.6%), the % cleaved (62% vs. 51%) and the transferable embryo production (5.4 vs. 3.8) was increased when 50 mg. PGF was administered in three divided doses rather than in two doses. In a second experiment doses of 15 mg., 30 mg. and 45 mg. (each administered as three divided doses 6 hours apart) all produced the same estrus response (95.6, 97.9 and 95%) and production of transferable embryos (4.9, 3.6 and 4.6). Three-times-a-day PGF reduced the time interval from treatment to the onset of estrus, but the time from PGF to estrus was not correlated with embryo production.