The effects of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocyte

The effect of serum obtained from a cow at the time of standing estrus (serum A), at ovulation (serum B), and at 24 h after ovulation (serum C) on the in vitro maturation and fertilization of bovine oocytes was examined. Of 144 (Group A), 159 (Group B), and 158 (Group C) oocytes, 77 (53.4%), 82 (51.6%) and 82 (51.9%) oocytes were characterized by expansion of cumulus cells, respectively. There was no significant difference in the effect of the three types of cow serum on the cumulus expansion (P < 0.05). Of 461 oocytes, 316 oocytes were cultured with sperm for fertilization, and 145 oocytes were cultured without sperm for evidence of parthenogenetic development. Of 56 (Group A), 56 (Group B), and 62 (Group C) oocytes with expanded cumulus cells, 19 (33.9%), 7 (12.5%), and 11 (17.7%) oocytes were cleaved, respectively, after exposed to the sperm for 24 h. There was a significant difference in the effect of the three types of cow serum on the fertilization rate (P < 0.05). A total of 145 oocytes was cultured in the absence of sperm and no evidence of parthenogenetic division was observed. The effect of the three types of serum obtained from the cow on the maturation of oocytes was not significant, but a significant difference did exist in the fertilization rate of oocytes. Cow serum obtained at the time of standing estrus had a beneficial effect on the fertilization rate of oocytes in vitro.     

Improved embryo yield and condition of donor ovaries in cows after PMSG superovulation with monoclonal anti-PMSG administered shortly after the preovulatory LH peak

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted. No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.     

Effect of castration on plasma luteinizing hormone in postpubertal boars

Two experiments were conducted to test the working hypothesis that mean plasma concentrations of luteinizing hormone (LH) increase as a result of an increase in the frequency and amplitude of the pulsatile releases of LH in postpubertal boars after removal of gonadal steroid hormones by castration. It was further hypothesized that these changes in secretion of LH would be the result of changes in sensitivity of the pituitary to gonadotropin releasing hormone (GnRH). In Experiment 1, plasma LH was monitored in 10 postpubertal crossbred boars (13 to 14 mo old and weighing 159 +/- 6.0 kg) at 12-min intervals for 6 h before and 1 h after GnRH (375 ng/kg of body weight) on Days -1, 7, 14, 21 and 29 relative to castration. In Experiment 2, plasma LH was monitored in four castrated and five intact postpubertal boars (11 to 12 mo old and weighing 150 +/- 5.1 kg) after each of three doses of GnRH (94, 188 and 375 ng/kg) were administered to each animal. Sample collection occurred 5 wk after castration. Mean LH and frequency of pulsatile releases of LH increased as a result of castration (P<0.0001), with changes evident by Day 7 after castration. However, the amplitude of the LH pulses increased minimally after castration (P<0.10). The response to exogenous GnRH increased throughout Experiment 1 (P<0.0001), even though the amplitude of the pulsatile releases of LH (response to endogenous GnRH) did not change. Castrated animals in Experiment 2 had a greater response of LH to GnRH stimulation than intact boars (P<0.05). The dose-response curve of castrated animals was not parallel (P<0.001) to that of intact boars, and indicated that sensitivity of the pituitary to GnRH had increased in the absence of gonadal steroids. Thus, the hypotheses stated above can be accepted with the exception that castration may have a minimal effect on LH pulse amplitude. Based on the results of these experiments, we suggest that gonadal steroid hormones modulate both the size of releasable stores of LH and pituitary sensitivity to GnRH in boars.     

Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics

Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender with 4% glycerol. Spermatozoal quality was assessed at four different points: at less than 15 min after collecting and before processing (Step 1); after centrifugation and just before freezing (Step 2); immediately after thawing less than 3 h after freezing (Step 3); and immediately after thawing 10 to 20 d after freezing (Step 4). Acrosome-specific monoclonal antibody detected differences (P <0.05) among steps and ejaculates within stallions. All parameters of spermatozoal motion, including the percentage of motile sperm, percentage of progressively motile sperm, curvilinear velocity, straight line velocity, linearity, amplitude of lateral head displacement, and radius of the average path for circularly swimming sperm, differed (P <0.05) among steps, and most of these parameters differed among ejaculates within a stallion and among stallions. For Steps 2 and 3, 62 and 37% of the sperm were motile, and 56 and 23% of all motile sperm had a curvilinear velocity of >100 mum/sec. Most damage to sperm occurred as a result of freezing-thawing, whereas centrifugation of sperm caused only minor damage.     

Comparisons of eight commercial on-farm milk progesterone tests

To evaluate eight commercial on-farm milk progesterone kits, milk samples (50 ml each of foremilk and postmilk strippings) were collected during the estrous cycle from 10 cycling Holstein cows for 24 consecutive days. Relative concentrations of progesterone were classified as low or high by comparison with standard progesterone samples supplied with each kit. The concentration of progesterone in each milk sample was determined by radioimmunoassay (RIA). Accuracy of classification into low or high levels by commercial tests was determined by the percentage of similarity with RIA values using discriminant analysis. Accuracy of the eight tests ranged from 89.0 to 98.9% for low progesterone, 74.8 to 85.6% for high progesterone, and 80.3 to 87.3% for all samples (n = 238). The percentage of fat in milk or an interaction of the percentage of milkfat by day of estrous cycle influenced commercial test results for all tests except Accufirm and Calfcheck. Progesterone levels, estimated by the test-kits, were low from 1.5 +/- 0.5 to 2.8 +/- 0.9 days before estrus (X +/- SEM) and until 4.0 +/- 0.6 to 5.9 +/- 1.3 days after estrus. These data support the principle that a single low progesterone sample cannot be used to determine proper timing of insemination. All eight commercial kits can be used to determine accurately the relative concentrations of progesterone in milk samples.     

Effect of early postpartum treatment with prostaglandin F2alpha on subsequent fertility in the dairy cow

Eighty Holstein dairy cows were treated with 25 mg of prostaglandin F2alpha (PGF2alpha) on Days 14 to 16 post calving. Eighty-four herd mates served as saline controls in the double blind study. The reproductive parameters used to measure fertility were mean days to first service on all cows and mean days open, first service conception rates and services per pregnancy on cows that became pregnant. Mean days to first service was similar in both groups (71.8 +/- 27, treated; 68.5 +/- 28.6, controls; P = 0.5352). The mean days open was 98.6 +/- 52.0 d for treated cows compared to 118.8 +/- 71.2 d for controls (P = 0.0680). First service pregnancy rates (treated, 41.3%; control, 35.7%) were not significantly different (P = 0.0630); however, the mean services per pregnancy (treated, 1.64; control, 2.33) were significantly (P = 0.0021) improved in the treated group. Ten cows in the treated group and twelve cows in the control group were diagnosed as having retained fetal membranes and/or metritis. For treated and control cows mean days to first service were 82.2 +/- 34.8 d and 82.8 +/- 58.7 d (P = 0.5652). Days open were 97.0 +/- 32.5 d and 133.4 +/-58.4 d (P = 0.3636); services per conception were 1.83 and 2.50 (P = 0.3178), respectively. In all treated cows reproductive parameters were similar whether cows had high serum progesterone ( > 1 ng/ml) or low serum progesterone ( < 1 ng/ml) on the treatment day. This study suggests that early postpartum treatment of lactating dairy cows with prostaglandin produces an improvement in fertility.     

The efficacy of estrus detection and fertility following synchronization with PGF2a or synchro - mate- B in Zebu cattle

Two experiments were carried out to assess the efficacy of estrus detection and fertility in Zebu cattle after synchronization with prostaglandin F2a or a progestagen. The first experiment compared estrus detection rates and fertility following insemination in 42 cows previously synchronized with either 25 mg of PGF2a or with a 6 mg of Norgestomet implant plus 5 mg i.m. of estradiol valerate (SMB). Differences were observed in the percentage of cows detected in estrus (54 vs 95%, respectively, P < 0.05), but not in fertility at the first synchronized estrus (26 vs 15%), nor in the detection rate and fertility at the subsequent estrous period (38 v 47%). The second experiment evaluated the efficacy of estrus detection at different time intervals in 30 cows, comparing estrus synchronized with PGF2a with the subsequent estrous period. The observation periods were continuous, day and night, for 100 h both after PGF2a treatment and from Day 18 of the treatment cycle (Period 1). In addition, the animals were administered PGF2a again on Day 10 of the second cycle and observed continuously from 0600 to 1800 h, and from Day 18 of the treatment cycle (Period 2). Finally, the same treatment regimen was used except that the observation was between 0600 to 0700 h and 1800 to 1900 h (Period 3). No differences were obtained in the percentage of cows detected in estrus in the synchronized and nonsynchronized groups (average 75%); however, accuracy in the detection of estrus in Period 3 differed in the nonsynchronized and synchronized estrus groups by 40% (P < 0.05) compared with the other two, more intense observation periods.     

Circulating concentrations of thyroid hormones in pregnant camels (Camelus dromedarius )

Blood samples from 16 female camels were collected at monthly intervals commencing from 60 d post. breeding until the last month of gestation. Two camels failed to conceive and two had unnoticed abortions. The average gestation period was 398+/-13 and 372+/-11 in camels bearing male and female fetus, respectively, with an overall mean of 383+/-9 d. Sera were analyzed for thyroxine (T(4)) and triiodothyronine (T(3)) by radioimmunoassay. Mean T(4) and T(3) concentrations varied from 76 to 116 ng/ml and 0.73 to 1.32 ng/ml, respectively, during various stages of gestation. In general, the T(4) and T(3) levels were higher during early pregnancy, with lowest values in the tenth month. T(4):T(3) ratio showed minor, nonsignificant fluctuations. Age of dam of sex of fetus had no effect on hormone levels. Similarly, hormone levels were not affected by failure of conception or by abortion.     

Dosage response evaluation of luprostiol administered to pregnant sows

Two trials were completed to investigate the effects of luprostiol in swine. The first trial was to evaluate parturition induced by various dosages of luprostiol compared with those of lutalyse or vehicle. Sows were assigned by random allotment to one of the following treatments on Day 112 of gestation: Group A, control (0 mg luprostiol); Group B (1.88 mg luprostiol); Group C (3.75 mg luprostiol); Group D (7.5 mg luprostiol); Group E (15 mg luprostiol); Group F (10 mg lutalyse). All prostaglandin-treated groups farrowed earlier than the controls (P<0.05), with Groups D (26.3 h), E (31.0 h) and F (25.8 h) having the shortest treatment-to-first-pig intervals, and Groups A (76.0 h), B (54.4 h) and C (40.0 h) having the longest intervals. Luprostiol-treated sows had the shortest farrowing time (P<0.05; range = 3.2 to 3.9 h). Significant differences were found for the time (min) between births: Group A (32.1), Group B (28.4), Group F (35.5) took longer than Group C (20.2), Group D (21.0) and Group E (21.6). In a second trial, 20 crossbred pregnant sows received either vehicle or luprostiol (7.5 mg) on Day 112 of gestation. Progesterone concentrations declined rapidly (P<0.05) in luprotiol treated females but were unchanged in control females during the 24-h collection period. The results of these trials show 7.5 mg luprostiol to be the most effective dose for inducing farrowing.     

A spectrophotometric procedure for the determination of objective measurements of equine spermatozoan motility

A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analyzed for the height (cm) and time (min) to the peak deflection. To standardize the procedure, a fixed number of cells (1x10(9)) were used to prepare suspensions of 300x10(6) cells/ml. Coefficients of variation for mean values obtained under these conditions after the evaluation of five ejaculates from a given stallion were estimated at between 10 and 12%. Correlations between swim-up measurements and computer-assisted semen analysis demonstrated that the percentage of motile cells and mean velocity (mum/sec) of motile cells influenced swim-up measurements. Described here is a simple and inexpensive procedure to determine objective measurements of spermatozoan motility that may have application in semen evaluation and fertility testing in the stallion.