A.H. Willemse

TRANSVAGINAL ULTRASOUND GUIDED FOLLICULAR ASPIRATION OF BOVINE OOCYTES

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.

Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a qualitative and quantitative milk progesterone test.

We compared three methods for diagnosing early pregnancy in cattle: 1) a trans-rectal ultrasound scan of the uterus, 2) a cow-side enzymeimmunoassay (EIA) milk progesterone test 3) a radioimmunoassay (RIA) milk progesterone test. Scanning of the uterus was performed in 148 cows. These cows were not detected in estrus before scanning, which took place between Days 21 and 33 after insemination (AI). A considerable difference was noted between the reliability of the scannings performed at an early stage (Days 21 to 25) and those performed at a later stage (Days 26 to 33). The sensitivity and specificity of the ultrasound examination between Days 21 and 25 were only 44.8% and 82.3%, respectively, but were 97.7% and 87.8% between Days 26 and 33, respectively. Milk samples were collected on the day of AI. (Day 0) and 21 days later. Samples that were positive in the EIA test always contained more than 1 ng/ml progesterone (P4); however, 20% of the negative EIA samples contained also more than 1 ng/ml P4. Only 59% of the animals showing a negative EIA test on Day 0 and a positive test on Day 21, indicating pregnancy, calved, while 16% of the cows with a negative test on Day 0 and Day 21, indicating nonpregnancy, turned out to be pregnant. Of the 82 animals with P4 levels lower than 1 ng/ml on Day 0 and higher than 1 ng/ml on Day 21, only 61.0% calved. All 14 cows with low levels both on Day 0 and Day 21, indicating nonpregnancy, were found to be not pregnant. The influence of both early embryonic death and the accumulation of intrauterine fluids on the accuracy of these tests are discussed.

Peripheral plasma concentrations of oestradiol, progesterone, cortisol, LH and prolactin during the oestrous cycle in the cow, with emphasis on the peri-oestrous period

Abstract Interrelationships of circulating hormone levels and their implications for follicular development were studied throughout the oestrous cycle with emphasis on the perioestrous period in heifers and cows. The oestradiol level showed a major peak (45 pmol/1) before and coinciding with oestrus, and a second peak (27 pmol/1) around day 5–6 (day 0: day of first standing oestrus); it was low during the luteal phase of the cycle when progesterone was higher than 14 nmol/1 from day −12 to day −2. Large antral follicles, which had developed during the luteal phase, did not secrete significant amounts of oestradiol, degenerated after luteolysis, and were replaced by a newly developing follicle which became preovulatory. Parallel with this development the oestradiol level increased from the onset of luteolysis to reach a plateau about 26 h before the onset of oestrus. The interval between the onset of luteolysis and the onset of oestrus was 58 h; luteolysis proceeded at a slower rate in heifers than in cows. At 4.6 h after the onset of oestrus the maximum of the LH surge was recorded; the LH surge appeared to be postponed in the period October–December in comparison to the period August–September. The maximum of the LH surge was higher in heifers (45 μg/l) than in cows (30 μg/l), but its duration was similar (8.0 h). The oestradiol level decreased significantly from 6 h after the maximum of the LH surge, and standing oestrus (duration 18 h) was terminated almost at the same time as the return to basal values of oestradiol. Cortisol and prolactin levels did not show a peak during the peri-oestrus period. Cortisol fluctuated irrespective of the stage of the oestrus cycle and prolactin was significantly higher during the luteal phase. The results of this study indicate that development of the preovulatory follicle starts in the cow at the onset of luteolysis, about 2.5 days before the preovulatory LH surge, and that oestradiol secretion by this follicle is possibly inhibited by the LH surge.

Evidence for prolactin as the main luteotrophic factor in the cyclic dog

The role of prolactin and LH in the control of the function of the corpus luteum in the dog was studied. Experiments were performed to interfere with the secretion of a) prolactin by administering a dopamine agonist and b) LH by desensitisation with a long-acting LHRH and by stimulation. Treatments with prolactin-lowering dosages of bromocriptine, (20 micrograms/kg body weight twice a day, orally; n = 8) which started between day 1-5 (n = 4) and day 20-24 (n = 4) of the luteal period resulted in a similar pattern of progesterone, concentration in peripheral blood in both groups. The progesterone release in the second half of the luteal period (13.1 +/- 1.8% (sem) of the progesterone release of the total luteal period) was significantly lower than in control dogs (24.7 +/- 2.2%). Treatment at about day 30 of the luteal period with LHRH CR (1.34 mg, intramuscularly; n = 3), which significantly suppressed the LH level, did not reduce the progesterone release in the second half of the luteal period, 21.3 +/- 4.7% compared to 24.7 +/- 2.2% in the control dogs. The endogenous LH peak resulting from treatment with LHRH had no effect on the progesterone concentration in the blood. It is concluded that prolactin is the main luteotrophic factor in the cyclic dog during the second half of the luteal period.

Evidence for the non-involvement of the uterus in the lifespan of the corpus luteum in the cyclic dog.

Progesterone levels in peripheral blood of dogs were analysed during the cycle in which hysterectomy (n = 5) or sham surgery (n = 3) was performed as well as during the cycle of dogs (n = 5) hysterectomized at least one year prior to this study; the data were compared with the findings in control dogs (n = 3). The averages of the duration of the luteal period observed in the three experimental groups were not significantly different from those of control dogs. Immediately after surgery, the progesterone level decreased from 25 to 50% of the presurgical level, but returned to presurgical level in about four days. Prolactin levels were elevated for about 30 h after surgery. Nevertheless, the averages of the mean prolactin levels for each animal during the luteal period of the experimental groups were not significantly different from those of control dogs. It is concluded that in the dog, the uterus is not involved in the lifespan of the cyclic corpus luteum.

Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up.

Repeated transvaginal ultrasound guided puncturing of visible follicles was performed for ovum pick-up (OPU) during Periods A and B, each of which lasted 3 mo. During Period A, 10 cows (A) were used in the study. Period B commenced 1 mo after Period A and two groups of animals were used. The first group (B1) consisted of 9 of 10 cows from Group A. The second experimental group of animals in Period B consisted of 11 cows (B2) which had not been submitted to previous puncture. During the study, all visible follicles larger than 3 mm were punctured and aspirated three times, on Day 3 or 4, Day 9 or 10 and Day 15 or 16 of the estrous cycle. The mean estrous cycle length (+/- SEM) after repeated follicle puncture did not differ among the three groups and was 22.3 +/- 0.4, 22.5 +/- 0.4 and 22.1 +/- 0.3 d for groups A, B1 and B2, respectively. The mean total number (+/- SEM) of punctured follicles per estrous cycle in Group A (13.1 +/- 0.5) was significantly larger than in Groups B1 (11.2 +/- 0.4) and B2 (11.6 +/- 0.4). The largest number of follicles punctured for ovum pick-up in all three groups was always on Day 3 or 4 of the estrous cycle: 4.9 +/- 0.3 follicles; the mean (+/- SEM) number of punctured follicles on Day 9 or 10 and Day 15 or 16 was significantly (P<0.05) lower: 3.4 +/- 0.2 and 3.9 +/- 0.2, respectively. In Period A, primarily 3- to 5-mm follicles were punctured per estrous cycle, while 6- to 10-mm follicles were predominantly punctured in Period B (P<0.05). Recovery rate of oocytes on Day 3 or 4, Day 9 or 10 and Day 15 or 16 were 53, 50 and 52%, respectively. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm.

Prolactin levels and the LH-response to synthetic LH-RH in the lactating sow

Abstract To determine the role of prolactin in the suppression of ovarian activity during lactation in the sow experiments were performed to investigate a possible inhibitory action of prolactin at the pituitary level. Therefore the LH-response to an intravenous injection of 25 μg synthetic LH-RH was measured in the 1st, 2nd and 3rd week of lactation. The compound was injected under high and low concentrations of prolactin in the peripheral blood, the latter achieved by removal of the piglets 6 h before administration of LH-RH. The results showed no difference in the effect of LH-RH injected at high or low prolactin levels. However, although the mean prolactin concentrations in the 1st, 2nd and 3rd week of lactation were similar, the results clearly demonstrated an increase in LH-response as lactation proceeds. The low responsiveness of the pituitary shortly post partum was also observed when the preparturient rise of prolactin was suppressed by treatment with bromoergocryptine. Injections of LH-RH in the last week of gestation given before and after the physiological increase of PRL, which occurred about 48 h before delivery, all showed low LH-response. It is obvious from the presented data that the LH-response to an intravenous injection of 25 μg LH-RH is in no way correlated with the prolactin levels at the time of treatment.

Plasma prolactin, progesterone and oestradiol-17β concentrations around parturition in the pig

Plasma concentrations of prolactin, progesterone and oestradiol-17β were measured by radioimmunoassay in samples taken from 2–15 days before until 1–4 days after spontaneous parturition in four sows and in one sow around prostaglandin F2α-induced parturition. Between Days −15 and −2 (Day 0 = parturition), prolactin concentrations in daily samples fluctuated somewhat, but exceeded 10 ng/ml only exceptionally. Plasma progesterone levels gradually declined or remained high until about 2 days before parturition. A final decrease of the progesterone concentrations coincided with distinct increases of the prolactin levels during the last 40 h of pregnancy. Maximal prolactin concentrations were measured before the onset of delivery of the piglets. Oestradiol-17β reached peak values around delivery. Prostaglandin F2α injection caused an immediate sharp increase of the prolactin concentration which lasted for about 6 h. During this period progesterone and oestradiol-17β concentrations did not change. A second elevation of prolactin levels was measured when progesterone finally decreased. Changes of prolactin concentrations found in this study were compared with those found in other domestic animals at the same reproductive stage.

Influence of Season and Parity on Several Reproductive Parameters in Nili-Ravi Buffaloes in Pakistan

Abstract In a study of effects of calving season and parity on the reproductive performance of the Nili-Ravi buffalo, 600 records of a large buffalo dairy farm complex under public sector management in Pakistan were analysed. The variables studied were parturition to first service interval (PS), first service to conception interval (SC), number of services per conception (NC), first service conception rate (FSCR), interval between successive services (SI) and calving interval (CI). In the analysis, parity was introduced at three levels: parity 1(A), parity 2, 3 and 4 (B) and parity 5 and above (C). Calving seasons were summer (May, June, July), autumn (August, September, October), winter (November, December, January) and spring (February, March, April). For all reproductive variables significant effects of season and parity were found. The interactions between season and partities were also significant except for the SI. Buffaloes which calved in summer and autumn showed a significantly better reproductive performance than their herdmates which calved in winter and spring. Parity effects indicated that PS, SC, SI and CI are significantly shorter in higher parity groups than in the first parity. It was observed that SI of 3 or 6 weeks are few, and a longer duration is common. It is concluded that a poor level of oestrus detection was the main contributing factor responsible for the longer SI. A few recommendations are made to curtail the negative influence of calving seasons on reproductive performance. A fertility status index is introduced to evaluate and compare fertility levels of buffalo herds.

Descriptive epidemiology and treatment of postpartum anestrus in dairy buffalo under small farm conditions

Three hundred and eighty-two preservice anestrous buffalo were examined between 60 and 90 d after parturition. These buffalo were randomly divided into two groups. Group I buffalo (n = 282) were examined per rectum and determinations of milk progesterone levels were carried out twice for each buffalo with a 10 d interval. Group 2 buffalo (n = 100) served as controls and did not receive the above examinations. Compatible results of palpation and milk progesterone levels revealed that the underlying causes of preservice anestrus in buffalo were substrus (206, 73.0%); true-anestrus (25, 8.9%); persistent corpus luteum (22, 7.8%); follicular cysts (15, 5.3%); luteal cysts (10, 3.6%); and pregnancy (4, 1.4%). Prostaglandin was used for treatment of subestrus (with active corpora lutea), persistent corpora lutea, and luteal cysts. Follicular cysts were treated with gonadotropin releasing hormone (GnRH) and true anestrus was treated with a progestogen-pregnant mare serum gonadotropin (PMSG) regimen. Success of the hormonal treatment was monitored by the determination of progesterone levels after the treatment. By this approach of diagnosis and treatment 62.7% (177282) preservice anestrous buffalo conceived, with a parturition to conception interval of 86.8 ± 13.5 d. During this period in the control group, only 25% of the buffalo (25100) conceived, with a parturition to conception interval of 88.1 ± 14.5 d. For the improvement of fertility in dairy buffalo under small farm conditions treatment is recommended.