Endocrinological evaluation of the induction of superovulation with PMSG in water buffalo (Bubalus bubalis)

Ten nonlactating buffalo were superovulated with 3000 IU PMSG. Luteolysis was induced with 500 mug Cloprostenol (PG) 60 and 72 h after PMSG. Five buffalo were alloted for natural mating and five were bred by artificial insemination 60 and 84 h after the first PG treatment. Since four buffalo developed pyometra, only 6 of 10 underwent embryo collection successfully 180 to 190 h after PG. Three buffalo yielded only one morula each, while the remaining three yielded a total of two, three and four morulae and/or blastocysts as well als zero, one and three unfertilized ova, respectively. Six of the ten buffalo were assigned to an intensive blood collection regimen. Mean concentrations of progesterone (ng/ml) increased from 1.9 at PMSG stimulation to 4.8 at induction of luteolysis and decreased to a nadir of 0.2 about 72 h after PG treatment. The preovulatory surge of LH occurred 36 +/- 9 h after PG and was low in magnitude (7.3 +/- 1.3 ng/ml). Stimulation of 3 to 12 follicles resulted in concentrations of estradiol-17beta exceeding 5 pg/ml within 48 h after PMSG treatment and reaching a maximum of 32 +/- 11 pg/ml about the time of the preovulatory surge. Only in two individuals did concentrations decrease below 5 pg/ml within the following 12 h. In the other four buffalo 3 to 10 unovulated structures remained palpable, secreting estradiol-17beta far exceeding the preovulatory concentrations. The fast appearing, low magnitude LH surges were key problems resulting from PMSG treatment. They caused unovulated endocrinologically active follicles. High estrogen levels during the early luteal period may activate subclinical uterine infections, which in turn may negatively affect embryonic development.     

Assessment of superovulatory responses in terms of palpable corpora lutea and embryo recovery using milk progesterone

Milk progesterone profiles were used to assess superovulatory responses in cyclic lactating buffalo (n = 9) in terms of the number of ovulations and the number of embryos recovered. All of the buffalo received a total of 30 ml of folltropin divided into morning and evening doses and spread over 5 days, beginning on Day 10 of the estrous cycle (day of expected estrus = Day 0). Milk samples for progesterone determination were collected on alternate days from all nine animals from Day 1 prior to the expected synchronized estrus to 5 days after flushing for embryo recovery. All animals were palpated per rectum 1 day prior to flushing in order to record the number of corpora lutea. Of an estimated 23 ovulations from the nine buffalo, only 12 embryos were recovered, of which one was an unfertilized oocyte. Milk progesterone profiles from individual buffalo suggested that a poor superovulatory response in terms of embryo recovery in some buffalo was caused by a failure to respond optimally to lutalyse treatment for the induction of estrus. It was hypothesized that ova trapping by the fimbriae of the fallopian tubes may not be efficent in this species especially in the superovulated ovaries.     

Embryo production and quality of Holstein heifers and cows supplemented with beta-carotene and tocopherol

The hypothesis was that the intramuscular injection (i.m.) of beta-carotene associated to tocopherol improves cow (n=86) and heifer (n=91) embryo production and quality. Time of estrus was synchronized in animals with an ear implant with 3 mg of norgestomet associated with an i.m. injection of 6 mg of norgestomet and 10mg of estradiol valerate (CRESTAR, Intervert International B.V., Boxmeer, Holland) and superovulated by 8 i.m. FSH/LHp injections (400 IU-heifers and 500 IU-cows) in decreasing concentrations at 12h intervals. Animals were inseminated 12 and 24h after observed onset of estrus and embryos recovered 7 days later. Animals were randomly allocated to one of three treatments: (1) vegetable oil vehicle (control), (2) 800 mg of beta-carotene and 500 mg of tocopherol (T800) and (3) 1200 mg of beta-carotene and 750 mg of tocopherol (T1200). Supplemental injections were given at the day norgestomet implants were inserted and at first superovulatory injection. An index (Embryo Quality Index or EQI) was proposed to more precisely evaluate embryo quality (excellent*1 + good*2 + regular*3 + poor*4 + degenerate*5 + unfertilized ova*5)/total. There was an interaction between physiological stage (heifer or cow) and treatment on EQI (P=0.01) and on the proportion of viable embryos (P=0.03), where both variables were improved in T1200 cows, but not in heifers. The average EQI for heifers and cows in control, T800 and T1200 were 2.6+/-0.3 and 3.6+/-0.3; 2.5+/-0.3 and 3.6+/-0.3; 2.9+/-0.3 and 2.7+/-0.3, respectively. The average total number of viable embryos was greater (P=0.01) in supplemented cows (3.5+/-1.1; 5.4+/-1.4 and 7.5+/-1.2 in control, T800 and T1200, respectively), but less (P=0.01) in heifers (7.5+/-1.2; 5.6+/-1.2 and 4.0+/-1.1 in control, T800 and T1200, respectively). Supplementation injections of beta-carotene associated to tocopherol improved embryo quality in superovulated Holstein cows, in the present experimental conditions and may be advantageous in similar embryo production systems. However, at dosages applied in the present experiment, this treatment should not be recommended for nulliparous heifers.     

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.     

Development of in vivo-matured porcine oocytes following intracytoplasmic sperm injection

The objective of this study was to assess the development of porcine ova fertilized by intracytoplasmic sperm injection (ICSI). Allyl trenbolone (Regumate) was used to synchronize estrus in 13 postpuberal gilts. Gilts were superovulated with pregnant mare serum gonadotropin and hCG. Ova were aspirated from 5- to 8-mm follicles at 36 h after hCG. Cumulus cells were removed by blunt dissection and pipetting in Beltsville embryo culture medium (BECM) supplemented with 0.1% hyaluronidase. Sperm were washed and resuspended in BECM + 8% polyvinylpyrrolidone. Ova (n = 237) that exhibited a polar body were centrifuged at 15 000 x g for 6 min and injected with a single spermatozoon. One hundred fifty-four ova were cultured in NCSU-23 medium in a 5% CO(2) in air environment for 168 h. Ova were fixed in acetic acid/ethanol and stained with 1% orcein. Sixty-nine ICSI ova were cultured for 24 h and transferred (mean = 23) to three recipients. Eighty-one ova (69%) that survived ICSI cleaved within 48 h. Thirty-eight percent (31/81) of these ova became blastocysts (mean +/- SEM = 24.7 +/- 1.1 cells). One recipient gave birth to three pigs. These results demonstrate that porcine embryos derived from ICSI can develop into live pigs.     

The effect of bovine pestivirus infection on the superovulatory response of Friesian heifers

The pathogenesis of reproductive loss associated with bovine pestivirus infection during the preovulatory period was investigated using superovulated heifers. Twenty-five Friesian heifers were selected and randomly assigned to either a control group (n = 12) which did not become infected or to a treatment group (n = 13) which became infected following intranasal instillation of 2 ml of serum inoculum containing 5.5 log(10) TCID(50)/ml non-cytopathic virus, 9 d prior to artificial insemination (AI). Transrectal ultrasonography was used to monitor follicular development and ovulation during the superovulatory period. Animals were superovulated using a standard protocol of twice-daily injections of FSH-P and then were inseminated twice commencing 12 h after the onset of estrus. The intensity of expression of estrus was higher in the control heifers than in the pestivirus-infected heifers. Of 13 pestivirus-infected heifers, only 3 heifers displayed standing estrus compared with that in the control group, in which 10 of 12 heifers exhibited standing estrus. The mean number of ova/embryos recovered from the control group heifers was 5.75 +/-2.31, of which 4.00 +/- 0.72 were evaluated as transferable quality embryos. In comparison, heifers in the pestivirus-infected group yielded only a mean of 0.60 +/-0.34 ova/embryos, of which 0.23 +/- 0.22 were transferable quality embryos. Based on ultrasonographic examination, 24 h after the first AI 82% of the presumptive ovulatory follicles had ovulated in the control group compared with an ovulation rate of only 17% in the treated group. The results of this experiment demonstrated that bovine pestivirus infection during the preovulatory period could adversely affect ovulation, thus leading to a significant reduction in the number of palpable corpora lutea and in the number and quality of embryos recovered.     

The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle

The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing device and estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group (TI; n=40) received a standard superovulation protocol with females of this group being between days 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B program containing 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were at day 0 (TII; n=30), between days 2 and 6 (TIII; n=30), days 7 and 12 (TIV; n=30), days 13 and 16 (TV; n=30) and days 17 and 20 (TVI; n=30) of the estrous cycle. Superovulation was induced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) at intervals of 12h. The donors received PGF2alpha (Cloprostenol) 48 h after beginning the treatment and CIDRs were removed 12h later. Artificial inseminations (AI) were performed 12 and 22 h after the initiation of estrus and embryos were collected 7 days after AI. The mean numbers (+/-S.E.M.) of total ova and embryos, viable (transferable) and degenerated embryos were 14.2+/-11.3, 7.4+/-6.9 and 3.2+/-3.5 (TI), 13.3+/-10.4, 7.1+/-6.2 and 3.3+/-4.3 (TII), 13.5+/-7.0, 8.1+/-6.7 and 2.3+/-3.0 (TIII), 17.4+/-9.9, 9.4+/-6.9 and 4.0+/-4.4 (TIV), 16.9+/-8.8, 9.8+/-8.1 and 2.7+/-2.5 (TV) and 13.0+/-7.8, 7.2+/-6.9 and 2.3+/-2.5 (TVI), with no significant differences (P>/=0.05) among groups. Pregnancy rates of 67.1% (TI; n=86/128), 60.8% (TII; n=59/97), 62.5% (TIII; n=73/115), 64.1% (TIV; n=84/131), 72.3% (TV; n=81/112) and 60.6% (TVI; n=63/104) were obtained with embryos transferred from these collections and did not differ significantly (P>/=0.05) among groups. The results of the present study allow us to conclude that a combination of steroid hormones may be used prior to superovulation in Nelore donors, at any stage of the estrous cycle without affecting the efficiency of embryo transfer programs.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Use of steroid hormone treatments prior to superovulation in Nelore donors.

The objective of this study was to evaluate the effectiveness of synchronization of follicular wave emergence using steroid hormone treatments in Nelore cows. Donors were placed into three groups. Those that were between days 9 and 12 of their cycle (estrus=day 0) formed the TI group (n=60), whilst those that were in any other stages of their estrus cycle constituted groups TII (n=60) and TIII (n=60). TI donors were submitted to a standard protocol of superovulation, however, TII and TIII donors were treated with the Syncro-Mate-B (SMB) or Controlled Internal Drug Releasing Device (CIDR-B) programs, respectively. Superovulation was induced with p-FSH, divided into eight decreasing doses at intervals of 12h. The donors received cloprostenol 48h after the beginning of the treatment and progestagens were removed 12h later. Artificial inseminations (AI) were done at 12 and 22h after the initiation of estrus and the embryo collections were done 7 days after AI. In the donors which displayed behavioral estrus, mean (+/-S.E.M.) total ova and viable (transferable) embryos were 15.8+/-1.4 and 8.3+/-1.0 (TI, n=56); 15.6+/-1.3 and 8.9+/-1.0 (TII, n=56); 17.3+/-1.0 and 9.9+/-0.9 (TIII, n=57), respectively, with no significant difference (P > or =0.05) among groups. In those animals that did not displayed behavioral estrus, the mean values of total ova and viable embryos were 3.5+/-1.6 and 0.7+/-0.5 (TI, n=4); 11.5+/-3.9 and 9.0+/-4.4 (TII, n=4); 8.7+/-5.0 and 5.0+/-2.9 (TIII, n=3), respectively, with no significant differences (P > or =0.05) among groups. Pregnancy rates of 62.2% (TI, n=235); 66.4% (TII, n=284) and 65.1% (TIII, n=244) were obtained with embryos transferred from these collections and did not differ significantly (P > or =0.05) among groups. It was concluded that the synchronization of the emergence of follicular waves in Nelore donors is usable and does not harm the efficiency of embryo transfer programs. In addition, in contrast to the standard superovulation protocol, this method permits the use of a large number of donors in a short time period, at any stage of the estrus cycle, minimizing the costs of embryo transfer.     

The effects of once or twice daily injections of pFSH on superovulatory response in heifers

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Superovulatory response of Sistani cattle to three different doses of FSH during winter and summer

Objective of the present study was to investigate the effect of season and dose of FSH on superovulatory responses in Iranian Bos indicus beef cattle (Sistani). Cyclic cows, in summer (n=16) and winter (n=16), were assigned randomly to three dose-treatment groups of 120 (n=10), 160 (n=12) and 200 (n=10) total mg of Folltropin-V with injections given twice daily for 4 days in decreasing doses. Estrous cycles were synchronized with two prostaglandin F2alpha injections given 14 days apart. From day 5 after the ensuing cycle, daily ovarian ultrasonography was conducted to determine emergence of the second follicular wave at which time superovulation was initiated. Relative humidity, environmental and rectal temperatures were measured at 08:00, 14:00 and 20:00 h for the 3 days before and 2 days after the estrus of superovulation. Non-surgical embryo recovery was performed on day 7 after estrus. The effects of season, dose, time of estrous expression and all two-way interactions were evaluated on superovulatory responses: total numbers of CL, unovulated follicles (10 mm), ova/embryo, transferable and non-transferable embryos. Season (summer or winter), doses of Folltropin-V (120, 160 or 200 mg NIH) and time of estrous expression (08:00, 14:00 or 20:00 h) did not affect the number of transferable embryos (3.1+/-0.58). When superovulatory estrus was detected at 08:00, a FSH dose effect was detected with the greatest numbers of CL (12.2+/-0.87) and total ova/embryos (12.2+/-1.46) occurring with 200 mg FSH (dosextime of estrous expression; P<0.01).     

Ovarian response and endocrine changes in buffalo superovulated at midluteal and late luteal stage of the estrous cycle: a preliminary report

A study was designed to determine whether superovulatory and endocrine responses in buffalo differ when gonadotropin treatment is initiated at midluteal and late luteal stages of the estrous cycle. Twenty-eight buffalo were randomized into 4 groups (A, B, C and D). Buffalo in Groups A and B (n = 8 each) were superovulated with Folltropin (total dose 25 mg) and Lutalyse. Treatments in Group A were initiated between Days 8 to 10 (midluteal group) and in Group B between Days 13 to 15 (late luteal group) of the estrous cycle. Buffalo in Groups C and D (n = 6 each) were not superovulated and served as controls. Blood samples from all groups of buffalo were collected daily for plasma progesterone and estradiol determinations. The number of corpora lutea (CL) and unovulated follicles was recorded (following per rectum palpations) 5 or 6 d post-estrus. Buffalo in Groups A and B exhibited estrus in larger proportions and earlier (49.33 +/- 3.82 h and 46.67 +/- 2.46 h, respectively) than the control Groups C and D (77.33 +/- 5.33 h and 78.0 +/- 3.83 h, respectively). Mean number of CL was higher in Group B (3.38 +/- 0.46) than in Group A (2.25 +/- 0.75), however,the difference was not significant (P > 0.05). Plasma progesterone concentrations on the day of treatment were higher in late luteal superovulated and control groups than in midluteal superovulated and control groups. In both Groups A and B progesterone levels were significantly related (r = 0.78,0.76; P < 0.05) to the number of CL palpated after the superovulatory estrus. Progesterone levels on the day of estimation of ovarian response were approximately 4 times higher in Groups A and B than in Groups C and D. Peak estradiol concentrations were approximately twice as high in superovulated groups as in control groups.     

Effect of estradiol supplementation on superovulation in Swamp buffalo

The effect of estradiol-17beta (E(2)) supplementation on superovulation with (PMSG) or (FSH) was investigated in Swamp buffalo. Sixty-eight buffalo were treated in seven groups. Group 1 served as control and was superovulated by standard PMSG or FSH treatment used in routine bovine embryo transfer protocols. Group 2 was superovulated by standard PMSG regimen plus two injections of E(2) at a 48 h interval beginning one day before the onset of gonadotropin treatment (short-term supplementation) for a total dosage of 2.5 mg E(2); Groups 3 and 4 received the same regimen as Group 2, but in doses of 5.0 and 7.5 mg E(2), respectively. Group 5 received the standard FSH regimen (40% LH). Group 6 received short-term E(2) (7.5 mg) supplementation of FSH-p. Group 7 was superovulated by standard FSH regimen (40% LH) plus three injections of E(2) at 48-72 h intervals beginning five days before the onset of gonadotropin treatment (long-term supplementation) for a total dosage of 7.5 mg E(2). The number of corpora lutea (CL) and follicles >/= 8 mm in diameter were recorded by palpation per rectum and after slaughter. The mean numbers of CL and follicles were 0.99, 5.8, 8.0, 10.6, 4.0, 3.9, 8.1 and 0.25, 6.8, 6.2, 6.2, 1.6, 0.0, 4.1 for Groups 1, 2, 3, 4, 5, 6, 7, respectively. In Group 7, the rates of nonsurgical and postmortem embryo recovery were 46 and 90.4%, respectively and 54.4% of the collected ova were fertilized. These results indicate the possibility of producing viable embryos in buffalo by using E(2) supplementation for the gonadotropin treatment.     

Factors affecting superovulation in heifers treated with PMSG

In this study we determined 1) if the immunoneutralization of PMSG affected the ovulatory response, the number of large follicles and embryo yield compared with that of PMSG alone or pFSH, and 2) whether the stage of the estrous cycle at which PMSG was injected affected the ovulatory response and yield of embryos in superovulated heifers. Estrus was synchronized in 99 (Experiment 1) and 71 (Experiment 2) heifers using prostaglandin F2alpha (PG) analogue, cloprostenol, given 11 d apart in replicate experiments over 2 yr. In Experiments 1 and 2, heifers were randomly allocated to 1 of 3 treatments (initiated at mid-cycle): Treatment 1--24 mg of pFSH (Folltropin) given twice daily for 4 d; Treatment 2--a single injection of 2000 IU PMSG; Treatment 3--2000 IU PMSG followed by 2000 IU of Neutra-PMSG at the time of first insemination. In Experiment 3, 116 heifers were given 2000 IU PMSG on Day 2 (n = 28), Day 3 (n = 27), Day 10 (n = 41) or Day 16 (n = 20) of the estrous cycle. The PG was given at 48 h (500 microg cloprostenol) and 60 h (250 microg cloprostenol) after the first gonadotropin treatment. Heifers were inseminated twice during estrus, and embryos were recovered on Day 7, following slaughter and graded for quality. The numbers of ovulations and large follicles (> or =10 mm) were also counted. There was no effect of treatment on ovulation rate in Experiment 1, but in Experiment 2 it was greater (P < 0.002) in heifers given PMSG (14.7 +/- 1.5) than pFSH (7.5 +/- 1.4) or PMSG-neutra-PMSG (8.7 +/- 1.5). The number of large follicles was higher following PMSG than pFSH treatment in Experiment 1, and it was higher (P < 0.004) in heifers given PMSG (5.5 +/- 0.8) than pFSH (1.12 +/- 0.7) or PMSG-neutra-PMSG (2.7 +/- 0.8) in Experiment 2. The use of Neutra-PMSG did not affect the numbers of embryos recovered or numbers of Grade 1 or 2 embryos, but it did decrease the number of Grade 3 embryos in both experiments. In Experiment 3, the ovulation rate decreased (P < 0.004) when PMSG was given on Day 3 (5.7 +/- 1.46) of the cycle rather than on Day 2 (12.3 +/- 1.64), Day 10 (13.4 +/- 1.45) or Day 16 (12.5 +/- 1.87). There was no effect of day of treatment on the numbers of large follicles. The mean numbers of embryos recovered were lower (P < 0.01) in heifers treated on Day 3 (2.1 +/- 0.67) than on Day 2 (6.8 +/- 1.0), Day 10 (6.4 +/- 0.86) or Day 16 (7.8 +/- 1.87). It is concluded that Neutra-PMSG given to heifers treated with PMSG did not improve embryo yield or quality and that treatment with PMSG early in the cycle can result in acceptable embryo yields provided sufficient time elapses between treatment and luteolysis.     

Alteration of follicular dynamics and superovulatory responses by gonadotropin releasing hormone and follicular puncture in cattle: a field trial

A field experiment was conducted to determine the influence of follicular alteration on superovulatory responses. Ultrasonography was performed once daily over 4 d prior to gonadotropin treatment (Day 0), on the day of estrus during superstimulation, and on the day of embryo collection to monitor follicular development. Animals were superstimulated between Days 8 and 12 of the estrous cycle. Follicular status was altered 2 d prior to initiation of superstimulation (Day 0) with GnRH (Cystorelin, 200 micrograms i.m.) administered with (GnRH-puncture group, n = 31) or without (GnRH-no puncture group, n = 52) concomitant removal of the largest follicle by follicular aspiration. Responses were compared with those of an untreated control group superovulated 8 to 12 d after estrus (n = 102). The proportion of animals with a high number (> or = 2) of large follicles (> = 7 mm) on Day 0 was lower (P < 0.001) in the 2 GnRH-treated groups than in the control group, while the increase in the number of medium size follicles (4 to 6 mm) on Day 0 was greater (P < 0.02) in the GnRH-puncture group. During superstimulation, the proportion of superovulatory cycles with a high follicular (> or = 10 follicles) response was similar in the control and GnRH-no puncture groups. Within the GnRH-treated animals, follicular and ovulatory responses were greater in the GnRH-puncture than in the GnRH-no puncture group (P < 0.001 to P < 0.02). Despite these changes in follicular and ovulatory responses, however, the mean number of embryos produced did not differ (P < 0.1) among treatments (4.3 +/- 0.4, 3.7 +/- 0.7, and 5.4 +/- 0.8 in control, GnRH-no puncture, and GnRH-puncture groups, respectively). This was due primarily to an increase in the mean numbers of unfertilized ova (P < 0.005) and in degenerated embryos (P < 0.06) in the GnRH-puncture group. Results indicate that the beneficial effects of treatment with GnRH and follicular puncture 2 d prior to superstimulation on follicular and ovulatory responses were limited by an increase in the number of unfertilized ova and degenerated embryos.     

Bull-specific effect on fertilization rate and viable embryo recovery in the superovulated buffalo (Bubalus bubalis)

This study was conducted to compare fertilization rate and viable embryo recovery rate in superovulated buffalo (n = 64) following insemination with semen from buffalo bulls (n = 5) having different fertility rates as determined by AI. Frozen-thawed semen from fertile bulls with similar post-thaw progressive motility and sperm morphology was used to inseminate buffalo at superovulatory estrus. Fertilization and viable embryo recovery rates differed among bulls, but this bull-specific effect was not related to the overall herd fertility rate as determined by AI in normal cyclic animals. These results indicate that individual bulls differ in their contribution to fertilization of superovulated donors and also to embryonic development, as determined by viable embryo recovery. Moreover, the results also suggest that buffalo bulls can be screened for optimal fertility and embryo recovery rates in superovulated donors. Further studies are warranted to ascertain the factors which contribute to such bull-specific effects.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.     

Effect of timing of prostaglandin PGF 2 alpha injection subsequent to embryo collection on the resumption of normal follicular development following superovulatory treatment in cattle

Nonlactating Holstein and Jersey cows (n = 24) were superovulated and ovarian follicular development was monitored by transrectal ultrasound during the period after embryo recovery. Luteolysis was induced by two injections of prostaglandin F(2)alpha (PGF; 25 mg Lutalyse; 12-h interval) at specific times after superovulatory induced estrus (Treatment 1, Day 9; Treatment 2, Day 12; Treatment 3, Day 17; Treatment 4, Day 25; superovulatory estrus = Day 0 of Cycle 1). Follicular development was monitored during Cycle 1 before and after PGF injection and continued through the ensuing estrous cycle (Cycle 2). Superovulation led to more than one embryo collected in 14 cows (mean = 8.71 embryos: positive superovulatory response [PSR] cows), while 10 cows were not successfully superovulated (mean = 0.1 embryo; negative superovulatory response [NSR] cows). These cows differed in terms of number of unovulated follicles detected at embryo collection (4.21 vs 17.2, PSR vs NSR) and plasma progesterone during the superovulatory estrous cycle (32.3 ng/ml PSR vs 8.6 ng/ml NSR). Follicular development during Cycle 1 started sooner in NSR than in PSR cows (day by class by response P<0.03) and was initiated on Days 11 to 12 in NSR cows and on Days 19 to 20 in PSR cows. Interval to estrus after PGF averaged 6.3 d. Cows having short intervals to estrus had follicles at the time of PGF injection. Treatment influenced the length of Cycle 1, but it did not affect the interval to estrus after PGF, the length of Cycle 2, or follicular development during Cycle 2. The results indicate that 1) the timing of PGF injection after embryo collection does not influence subsequent follicular populations, 2) elongated estrous cycles and intervals to estrus after PGF in superovulated cattle are a function of decreased follicular activity, and 3) the presence of numerous corpora lutea and not the superovulatory treatment, per se, seem to attenuate follicular growth.     

Effects of GnRH on superovulated cattle

Gonadotropin releasing hormone (GnRH) was given to 109 cows and heifers during the course of 224 superovulations. Follicle stimulating hormone (FSH) was administered twice daily (5 or 6 mg) for 3.5 to 4 days beginning on any of Days 9 to 14 of the estrous cycle; prostaglandin (45 mg PGF(2)alpha or 750 ug cloprostenol) was given in a split dose on the fourth day. Donor cows and heifers were placed into four groups according to previous superovulation treatments, which consisted of one to three treatments or of no previous treatment. Every other cow or heifer within each of the four subgroups was treated with GnRH (200 mug i.m.) at standing estrus. Only donors that exhibited estrus within 32 to 72 h after the first prostaglandin treatment were used in the study. Animals were inseminated artificially 12 and 24 h after standing estrus was first observed. No differences were noted in the number of ovulations, total ova or transferable embryos recovered from the GnRH or control groups; however, two interactions were detected. Cows given GnRH had fewer palpable corpora lutea than control cows (P < 0.05), but this difference was not seen in heifers. The second interaction was that GnRH seemed to depress ovulation rate in donors not previously superovulated, but this effect was not observed with subsequent superovulations. Cows yielded more total ova than heifers (P < 0.01). There was no difference in return to estrus between GnRH and control groups after a second prostaglandin treatment at the time of embryo recovery. Most donors within each group resumed cycling between 5 and 12 d after embryo recovery.