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.     

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.     

Endocrine profiles and embryo quality in superovulated Japanese Black cattle

The relationships between plasma concentrations of progesterone (P(4)), estradiol-17beta (E(2)) and lutenizing hormone (LH) and embryo yield and quality were examined in 40 superovulated Japanese Black cattle. The results indicated that the ovarian function, especially the function of corpus luteum on the first treatment day, is an important factor for reliable superovulation in cattle. The levels of plasma E(2) and LH at estrus were related to embryo yield and quality.     

Plasma progesterone profiles and embryo quality in superovulated Japanese Black cattle

The relationship between plasma progesterone (P(4)) levels before and after superovulation treatment and embryo yield and quality was examined in Japanese Black cattle. It is concluded that a plasma P(4) level at the start of follicle stimulating hormone (FSH) injections is related to embryo quality. The P(4) level of over 3.0 ng/ml appears to be useful in preselection of donors when we want to get more than 8 normal (transferable) embryos on the average.     

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.     

Plasma progesterone levels in superovulated cattle in relation to hatched embryo sizes,ovulation rates,and premature regression of corpora lutea

Progesterone concentrations were measured in peripheral plasma of 62 cattle treated with PMSG. There was good correlation (r = 0.92) between a single day-10 value and the sum of daily values up to day 12 (19 animals) or day 13 (17 animals). The day-10 progesterone level was correlated strongly with ovulation rate (r = 0.76; 55 animals) but to a negligible extent with the sizes of 306 day-13 embryos from 47 donors (r = 0.17). Premature regression of corpora lutea, encountered in 14 (7.3%) of 191 flushed donors, began between days 5 and 8 and was reflected in the progesterone profiles of seven animals that were serially sampled.     

Recovery rates and embryo quality following dominant follicle ablation in superovulated cattle

To determine the association between dominant follicle ablation and the outcome of a superovulatory regimen, two data sets were constructed from records of 171 recoveries from non-ablated cows and 1214 recoveries from cows that underwent follicular ablation prior to FSH treatment. Data set 1 included all cows with 2 or more records (n = 1385). Data set 2 included paired data for 87 cows which had at least 2 records of both ablated and non-ablated superovulatory attempts. Dominant follicle ablation was performed by use of transvaginal, ultrasound guided aspiration 48 hr prior to the start of FSH. The same FSH protocols were used for both ablated and non-ablated cows. For all cows (data set 1), more total ova/embryos were recovered from the ablation group (12.1+/-0.3 vs 10.5+/-0.8; P=0.06). This difference could be accounted for by greater numbers of non-transferable embryos in the ablation group (6.5+/-0.2 vs 5.3+/-0.6; P>0.01). For the paired data (data set 2), greater numbers of total ova/embryos recovered from the ablation group (12.8+/-1.0 vs 9.7+/-0.7; P=0.01) could also be accounted for by higher numbers of nontransferable embryos in this group (7.8+/-0.8 vs 4.5+/-0.4; P>0.01). There were no differences between groups for high quality embryos, percent cows producing no ova/embryos or percent cows producing no transferable embryos. These data support the premise that synchronization of follicular waves following dominant follicle ablation increases total ova/embryo output. However, the additional embryos were primarily nontransferable thereby negating potential economic gains.     

Effect of the luteinizing hormone on embryo production in superovulated rabbit does

For most domestic animals, the responses to superovulation treatments are not controlled as a consequence of the lack of knowledge on exogenous gonadotrophins effects on the ovarian function. The role of luteinizing hormone (LH) on the number and quality of embryos produced was evaluated on rabbit does superovulated with porcine FSH (pFSH). Parameters of embryos recovery, in vitro and in vivo embryo development rates after freezing/thawing were compared. We used three experimental groups: (1) control group without superovulation treatment, (2) "pFSH+pLH" and (3) "pFSH" groups where females were treated with pFSH, respectively, with (20%) or without (0%) porcine LH supplementation. The number of corpora lutea and the number of embryos produced were significantly higher (p<0.001) in superovulated does than in control group (27.1, 26.7 versus 11.9 corpora lutea and 20.3, 21.2 versus 9.6 embryos produced for pFSH+pLH, pFSH and control group, respectively). However, both gonadotrophins administrations (groups 2 and 3) led to defaults of ovulation when compared with untreated does. No significant difference was observed between the number and quality of the embryos produced by does treated with pFSH+pLH or with pFSH alone. Moreover, we observed no significant difference between results of in vivo and in vitro viability assays after thawing. We concluded that pFSH alone seems to be sufficient to stimulate the follicles growth and that exogenous pLH administrated has no effect on the quantity and quality of embryos. Further studies are needed to evaluate the hormonal patterns before and after the gonadotrophins injections in the rabbit species.     

Transferable embryo recovery rates following different insemination schedules in superovulated beef cattle

The objective of this study was to evaluate the transferable embryo recovery rates from superovulated donor cattle after different artificial insemination (AI) schedules. Sixty mixed-breed crossbred females were administered follicle stimulating hormone (FSH) and prostaglandin F(2)alpha (PGF(2)alpha) to induce a superovulatory response. At standing estrus, donor females were randomly allotted to one of five treatment groups for AI. Donors were inseminated with two units of high-quality or low-quality frozen semen at 12, 24, 36, or 48 h after the onset of estrus in treatment Groups I, II, III, and IV, respectively, or inseminated with two units at 12, 24, 36, and 48 h (eight units/donor) in control Group V. Donor females inseminated once at either 12 or 24 h after the onset of estrus did not differ from donors inseminated in Group V in overall fertilization and transferable embryo recovery rates. The highest fertilization rate (89.5%) and transferable embryo recovery rate (74.9%) per donor resulted when AI was performed with high-quality semen at 24 h after the onset of estrus. These findings indicate that repeated insemination of superovulated beef cattle is not necessary to attain optimal fertilization rates and production of transferable quality embryos in beef cattle.     

Donor category and seasonal climate associated with embryo production and survival in multiple ovulation and embryo transfer programs in Holstein cattle

The present study investigated the effect of Holstein donor category (cows vs. heifers) and climate variation (hot vs. cooler season) on the efficiency of in vivo embryo production programs as well as embryo survival after transferred to Holstein recipient cows. A total of 1562 multiple ovulation (MO) procedures (cows: n = 609, and heifers: n = 953) and 4076 embryo transfers (ETs) performed in two dairy herds were evaluated. Donor cows had greater number of CLs (10.6 ± 0.6 vs. 7.5 ± 0.4; P < 0.0001) and ova/embryos recovered (7.6 ± 0.6 vs. 4.6 ± 0.4; P < 0.0001) compared with donor heifers. However, fertilization rate (47.9 vs. 82.4%; P < 0.0001) and proportion of transferable embryos (31.5 vs. 67.4%; P < 0.0001) were lower in donor cows than heifers, respectively. Regardless of donor category, the proportion of freezable embryos was less (P < 0.001) during hot season than in cooler season (21.4 vs. 32.8%). However, greater decline in the proportion of freezable embryos during the hot season was observed in cows (21.7 vs. 10.7%) compared with heifers (46.2 vs. 38.1%; P = 0.01). In contrast, the season on which the embryo was produced (hot or cool) did not affect pregnancy rate on Day 31 (30.5 vs. 31.7%; P = 0.45) and 45 (25.3 vs. 25.1%; P = 0.64) of pregnancy. Regardless of the season in which the embryos were produced, embryonic survival after transferring embryos retrieved from donor cows was greater on Days 31 (36.0 vs. 30.7%; P = 0.001) and 45 (28.3 vs. 23.1%; P = 0.001) of pregnancy when compared with embryos from donor heifers. In conclusion, MO embryo production efficiency decreased during the hot seasons both in cows and heifers; however, the decline was more pronounced in donor cows. Regardless of the embryo source, similar pregnancy rate was observed in the recipient that received embryos produced during the hot and cooler seasons. Curiously, embryos originating from donor cows had higher embryonic survival when transferred to recipient cows than embryos originating from heifers.     

Embryo production from superovulated cattle following insemination of sexed sperm

Two trials were conducted to ascertain fertilization rate, embryo quality and numbers of transferable embryos in superovulated heifers and cows inseminated with sexed sperm. Inseminates contained 2 x 10(6), 10 x 10(6) or 20 x 10(6) total sperm enriched for the X- or Y-chromosome ( approximately 90%) by flow cytometry/cell sorting. Non-sexed inseminates contained 40 x 10(6) total sperm. Donors in each trial were allocated to one of each of the bulls included in that study. Each donor was inseminated with frozen/thawed sperm from the same bull for each treatment in successive courses of superstimulation with twice daily i.m. injections of FSH for 4 d. Heifers and cows were inseminated 12 and 24 h after visually observed standing estrus in Trial 1. In Trial 2, a single timed inseminate was used 70-72 h following PGF(2alpha). Ova/embryos were collected non-surgically 7-7.5 d after insemination. In both trials, fewer ova were fertilized with sexed versus non-sexed treatments and with 2 x 10(6) sexed sperm compared to higher doses (P < 0.05). However, insemination of 20 x 10(6) total sexed sperm of >or=90% purity resulted in similar numbers of transferable embryos of the desired sex compared to that for non-sexed sperm.     

Plasma progesterone concentration in relation to ovulation rate and embryo yield in Chios ewes superovulated with PMSG

The main purpose of this work was to investigate the relationship between plasma progesterone concentration and the number of ovulations and/or the number of embryos collected from Chios ewes induced to superovulate with various doses of PMSG.The oestrous cycles of the animals were synchronized by means of MAP intravaginal sponges for 14 days and PMSG was injected i.m. (1500 IU, Group 1; 1000 IU, Group 2; 750 IU, Group 3; 500 IU, Group 4; 0 IU, Group 5) at the time of sponge withdrawal. Seven days after sponge removal and 5 days after mating, mid-ventral laparotomy was performed and the uterine horns and/or oviducts were flushed. The number and diameter of corpora lutea (CL), the number of large (diameter > 0.5 cm) anovulated follicles and the total ovarian response (TOR = CL + large anovulated follicles) were recorded. The embryos were examined under a dissecting microscope and evaluated according to morphological criteria. Blood samples were collected once daily for 4 days starting on the day of sponge withdrawal. One more sample was taken on the day of embryo collection. Progesterone concentration was determined using a conventional ELISA.A significant positive correlation was found between plasma progesterone concentration and number of corpora lutea (r = 0.61, P < 0.001), total diameter of corpora lutea (r = 0.63, P < 0.001), total ovarian response (r = 0.63, P < 0.001), number of eggs (r = 0.51, P < 0.001), number of embryos (r = 0.43, P < 0.001) and number of transferable embryos (r = 0.36, P < 0.01) collected per ewe treated. A negative relation between progesterone concentration (≥ 2 ng ml⁻¹) at the beginning of oestrus and number of corpora lutea (CL) was observed. The investigation of the relationship between ovulation rate and plasma progesterone concentration on the day of embryo collection resulted in the calculation of a formula for the prediction of the response of Chios sheep after superovulation with the specific hormonal regimen.     

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.     

Studies of the pathogenesis of bovine pestivirus-induced ovarian dysfunction in superovulated dairy cattle

Two experiments (Experiment I, n=12 Holstein-Friesian heifers; Experiment II, n=8 Jersey cows) were conducted to investigate the pathogenesis of bovine pestivirus-induced ovarian dysfunction in cattle. In both experiments the cattle were superovulated with twice daily injections of a porcine pituitary extract preparation of follicle stimulating hormone (FSH-P), for 4 days commencing on Day 10+/-2 after a presynchronised oestrus. The heifers received a total dose of 30 mg and the cows 32 mg of FSH-P. Prostaglandin F(2alpha) (PGF(2alpha)) was administered 48 h after commencement of superovulation and all cattle were artificially inseminated (AI) between 48 and 66h after PGF(2alpha) treatment. In both experiments bovine pestivirus seronegative cattle (Experiment I, n=6; Experiment II, n=4) were inoculated intranasally with an Australian strain of non-cytopathogenic bovine pestivirus (bovine viral diarrhoea virus Type 1) 9 days prior to AI. Bovine pestivirus infection was confirmed by seroconversion and/or virus isolation in all of the inoculated cattle, consistent with a viremia occurring approximately between Day 5 prior to AI and the day of AI. Ovarian function was monitored in both experiments by daily transrectal ultrasonography and strategic blood sampling to determine progesterone, oestradiol-17beta, luteinising hormone (LH) and cortisol profiles. Non-surgical ova/embryo recovery was performed on Day 7 after AI. In Experiment II half the cattle were slaughtered on Day 2 and the remainder on Day 8 after AI, and the ovaries submitted for gross and histopathological examination including immunohistochemistry to demonstrate the presence of bovine pestivirus antigen. In both studies, comparisons were made between infected and confirmed uninfected (control) animals. Overall the bovine pestivirus infected cattle had significantly lower (P<0.05) ova/embryo recovery rates compared to the control cattle. There was evidence of either an absence (partial or complete) of a preovulatory LH surge or delay in timing of the LH peak in the majority (90%) of infected heifers and cows, and histologically, there was evidence of non-suppurative oophoritis with necrosis of granulosa cells and the oocyte in follicles from the infected cows. By contrast only 20% of the control heifers and cows had evidence of absence of a pre-ovulatory LH surge. These experiments collectively demonstrate that bovine pestivirus infection during the period of final growth of preovulatory follicles may result in varying degrees of necrosis of the granulosa cells with subsequent negative effects on oestradiol-17beta secretion which in turn negatively affects the magnitude and/or timing of the preovulatory LH surge.     

Active immunization with a synthetic fragment of pig inhibin alpha-subunit increases ovulation rate and embryo production in superovulated ewes but season affects its efficiency.

Two experiments were designed to determine the effects of active immunization against one of two synthetic peptides from humans (inhibin-like peptide) or pigs (inhibin alpha-subunit) on antibody titres, ovulation rate and embryo production in ewes superovulated with 16 U ovine FSH. In Expt 1, during the breeding season, 30 ewes were subdivided into three groups: group I served as the non-immunized control; group II was immunized against inhibin-like peptide (100 micrograms inhibin-like peptide equivalent, followed by three booster injections); group III was immunized against pig inhibin alpha-subunit conjugated to human serum albumin (96 micrograms for the primary administration and 46 micrograms for the booster). In Expt 2, the efficiency of immunization against pig inhibin alpha-subunit on ovarian response and embryo production was evaluated during the non-breeding season in two groups of ewes (n = 12): group IV was a non-immunized control; Group V was immunized against pig inhibin alpha-subunit. During the breeding season, the ewes immunized against pig inhibin alpha-subunit showed higher antibody titres compared with the group immunized against inhibin-like peptide (P < 0.01) and a significant increase in ovulation rate (12.1) compared with both the control (5.0; P < 0.05) and the inhibin-like peptide-immunized group (3.1; P < 0.01). Immunization against pig inhibin alpha-subunit increased transferable embryo yield 4.5-fold (6.7 versus 1.5; P < 0.01) and improved embryo quality (94.6 versus 40.6%; P < 0.01). During the non-breeding season, immunization against pig inhibin alpha-subunit enhanced ovulation rate from 2.6 in the controls to 9.4 (P < 0.01) but did not affect transferable embryo production (3.9 versus 2.1; P > 0.05) and significantly lowered their quality (54.1 versus 100%; P < 0.01). In conclusion, active immunization against pig inhibin alpha-subunit can improve superovulatory response during the breeding season, while it appears to be unable to increase embryo yield during the seasonal anoestrus.