Effects of estradiol-17 beta and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet.

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2-3 months postpartum were used to investigate the effect of addition of estradiol-17 beta and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production. The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 micrograms cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17 beta given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.

[Control of terminal follicular maturation during the follicular phase in domestic mammals]. / Contrôle de la maturation terminale des follicules au cours de la phase folliculaire chez les mammifères domestiques.

Terminal follicular maturation in the ovine and the bovine species involves growth and differentiation processes in follicles between 1-2 mm diameter and the preovulatory stage. During this maturation, the follicle acquires the ability to ovulate and the oocyte becomes able to be fertilized and to develop after fertilization. Selection of ovulatory follicles results from the integration of different parameters such as the circulating levels of gonadotropins, the structure of follicular populations and the sensitivity of the hypothalamo-pituitary axis to ovarian hormones. Differences between follicles for FSH and LH responsiveness can be amplified by paracrine intrafollicular regulations. These mechanisms are probably determinant for selection of ovulatory follicles.

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.

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.

Are antibodies responsible for a decreased superovulatory response in goats which have been treated repeatedly with porcine follicle-stimulating hormone?

Repeated administration of xenogenic gonadotropins in human or animal species may be responsible for antibody production and refractoriness. An experiment was conducted in which goats were treated with porcine FSH (p-FSH) at 6-week intervals for a period of 7 months. A sensitive radioimmunoassay (RIA) was used to detect antibodies to p-FSH in plasma samples taken at short-term intervals during a 7-month period. Antibodies appeared after the first injection, and levels increased following booster injections. A high correlation rate existed between antibody level and superovulatory response. Refractoriness in goats was associated with a high level of antibodies.

Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cell monolayers.

Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cells was assessed1) by the ability of spermatozoa to fertilize bovine oocytes in vitro and2) by exposure to lysophosphatidylcholine (LC) to induce acrosome reaction in the capacitated spermatozoa. When spermatozoa were incubated on bovine epithelial oviduct cells in B2 medium supplemented with 10% estrous cow serum (ECS) and then exposed to 100 microg/ml LC for 15 minutes, the percentage of acrosome reaction induced increased in a time-dependent course, reaching a plateau after 6 hours. Inversely, when spermatozoa were incubated in B2+10% ECS alone, the percentage of acrosome reaction induced by LC didn't fluctuate. The in vitro fertilization rate obtained after incubation of spermatozoa during 6 hours on bovine epithelial oviduct cells in B2+10% ECS medium was on average 75% for both the preovulated and ovulated oocytes. The developmental stages observed 18 hours after male and female gamete co-culture were similar to those obtained after in vivo fertilization. This study suggests that incubation of fresh bovine spermatozoa on bovine oviduct epithelial cell monolayers during 6 hours is an efficient method, and one that is close to in vivo capacitation.

Survival of ovine embryos stored at 4 degrees C for 24 hours.

This study was conducted to ascertain if sheep embryos collected for transfer can be stored for short periods without freezing to allow for international transport. Of twelve Finnish Landrace ewes treated with equine follicle stimulating hormone (FSH), eleven ewes ovulated with a mean of 9.1 +/- 4.3 (SD) corpora lutea. Recovery rate from the nine ewes with normal corpora lutea was 68 +/- 27%, providing 61 morulae which were then cooled to 4 C and stored for 24 h while transporting them from Scotland to France. Romanov recipients received either 4 (n = 14) or 5 (n = 1) of these morulae. Fourteen of the recipients lambed, with a mean lambing rate of 2.1 +/- 0.8, representing 48.3% of embryos transferred. Cooling of embryos to 4 C and storing them in ovum culture medium for 24 h at 4 C may be a valuable technique for the handling and short-term storage of embryos.

Improvement of survival rate of frozen cattle blastocysts after transfer with trophoblastic vesicles.

An experiment was conducted to determine if the loss of viability due to deep freezing could be overcome by addition of trophoblastic tissue to the embryo at transfer time. Forty-nine recipient heifers in a cotransfer group each received one frozen blastocyst + two frozen trophoblastic vesicles. The confirmed pregnancy rates by Day 45, 60, and 90 were 73, 61, and 57%, respectively. In a control group of 53 recipients that received only a frozen blastocyst, pregnancy rates for the same periods were 43, 42, and 40%, respectively. The difference between groups was highly significant by Day 45. The addition of trophoblastic vesicles to frozen embryos contributed to luteal maintenance in recipients and likely magnified the intensity of embryonic signals resulting in improved pregnancy rates.

Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue transmission.

In order to import non-seasonal Creole goats from the Carribean to Europe for an experimental purpose, thirty Creole goats were treated with 10 mg of FSH; embryos were collected at slaughter, washed and deep frozen. After rapid thawing, they were reimplanted surgically into European dairy goats. Twenty-four females ovulated but only 17 of the ovulating females had functional corpora lutea (CL) at collection. Ovulation rate (CL goat ) and recovery rate (embryo CL ) were 13.8 and 78% for females with functional CL. Of 191 embryonic structures collected, 79% were considered suitable for deep freezing: 23% were young blastocysts, 47% were expanded blastocysts, and 30% were zona-pellucida (zp)-free and zp-damaged embryos. Seventy-eight embryos were thawed and 63 were reimplanted. Sixty-eight percent of the recipient females delivered 19 kids. The percentage of kids born relative to good-quality re-implanted embryos was higher for zp-free embryos (64%) than for young and expanded blastocyts (36%). Forty-seven percent of the donor females had strong positive serological reactions for bluetongue virus antibodies against serotypes 6 and 14. However, no recipient goats or newborn kids were positive. Virus isolation attempts on the collection media and last embryo washes were negative.

Quick freezing of rat embryos.

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).

Induction of superovulation in cyclic heifers: The inhibitory effect of large doses of PMSG.

In two different experiments, superovulation was attempted with a PMSG-PG treatment; a bovine anti-PMSG serum was injected at estrus. After 2500, 5000 and 7500 IU of PMSG injected during the luteal phase, the mean ovulation rates were respectively 16.2 +/- 7.7, 3.2 +/- 2.1, and 1.4 +/- 0.6 in the first experiment (17 heifers) and 18.3 +/- 12.6, 8.5 +/- 8.2, and 2.2 +/- 2.3 in the second (19 heifers). The estradiol-17beta and progesterone patterns and the observations of the ovaries on the day of estrus (Day 0) by ultrasonic echography and on Day 8 by endoscopy show that the ovaries were highly stimulated and suggest that the inhibition observed with the largest doses reflects the absence of the preovulatory LH discharge or its effect.

Changes in milk and plasma concentrations of progesterone in cows after treatment to induce superovulation and their relationships with number of ovulations and of embryos collected.

Progesterone concentrations in the milk of 86 Friesian cows induced to superovulate with an FSH-Cloprostenol treatment were studied daily from the day of estrus (D 0) to Day 7 (D 7). From D 2, a significant correlation between progesterone concentrations and ovulation rate was observed. Such a relationship was also observed beginning to D 3 between progesterone concentrations and the number of embryos recovered. No relationship was found between progesterone content and the number of viable embryos. For 30 of these cows, progesterone concentrations in blood plasma were also studied. The hormonal patterns in plasma and milk were similar but quantitative relationships were demonstrated earlier for progesterone in plasma than for progesterone in milk. It is concluded that relationships between milk progesterone concentrations and ovarian responses to a superovulatory treatment exist and could be of interest in embryo transfer programs in when predicting the number of embryos to be recovered.

Effect of injection time of anti-PMSG antiserum on ovulation rate and quality of embryos in superovulated cows.

Eighteen cows were superovulated by injecting 3000 IU of PMSG during the luteal phase, followed 48h later with an injection of Estrumate. They were then placed in a control group or were given anti-PMSG antiserum at either 12h or 24h after the onset of oestrus. Sixteen of these animals were used for the same experiment five months later. The results were pooled because they were not significantly different between the two treatment periods. The timing of the injection of anti-PMSG antiserum, either 12h (11 cows) or 24h (12 cows) after the onset of oestrus, did not significantly affect the ovulation rate, the number of embryos collected or the number of good embryos. The antiserum significantly increased the number of good embryos but did not affect the ovulation rate or embryo recovery. It is concluded that even with a moderate dose of PMSG, the use of anti-PMSG at 12h or 24h after the beginning of oestrus improves the quality of embryos. The mean number of embryos to be transferred (5.5) is in the range of those obtained after the FSH treatments, but the procedure required only three injections compared with nine for the FSH treatment.

Comparison of two methods for one-step in-straw thawing and direct transfer of cattle blastocysts.

The aim of this experiment was to evaluate the feasibility of dilution of the cryoprotectant by a sucrose solution in the straw followed by direct transfer without any selection of the embryos. A comparison was made between the method described by Renard et al. (7) and a method slightly modified from that published by Leibo (8). The feasibility is proved by a mean pregnancy rate of 41.4%. Although the difference was not statistically significant, the method described by Renard et al. (7) led to a higher pregnancy rate (44.7%) than that of Leibo (8) (38.5%).

Two-step freezing of cattle blastocysts with dimethylsulfoxyde (DMSO) or glycerol.

Sixty five cattle blastocysts were frozen by the so-called two-step freezing method: The samples were seeded at -7 degrees C and then directly brought at -30 degrees C for 30 minutes before being taken into liquid nitrogen. Results in terms of survival rates at thawing and after short term cultures were compared to two controlled linear cooling rate procedures (i.e. 0.3 degrees C/min and 1.3 degrees C/min). The results demonstrate that: 1) two-step freezing yielded approximately the same survival rate as the two others techniques and 2) Glycerol yielded better survival rates than DMSO treatments (56 vs 31% after 24 hours in culture).

Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicles at the end of the bovine estrous cycle.

In cows, electrocauterization of large follicles at the end of the luteal phase, lengthened the life span of corpora lutea. Injection of 5 mg of estradiol valerate or of 1 mg of an analogue of prostaglandin F(2alpha) induced luteolysis; however, the injection of estrogen was associated with precocious estrus without either ovulation or corpus luteum growth. Injection of both estradiol valerate and prostaglandin analogue gave the same results as estradiol valerate alone. Deferred luteolysis, observed after electrocauterization of large follicles, seemed to be due to the withdrawal of estrogens and the consequent lack of prostaglandin F(2alpha) production.

The non-luteolytic effect of prostaglandin F(2)alpha at the beginning of the bovine oestrous cycle: the role of oestrogen?

After the preovulatory gonadotrophin surge, antral follicles ovulate or become atretic; whatever their evolution, they stop secreting oestradiol. Since it was demonstrated that oestrogens were necessary for luteolysis to occur after PGF(2)alpha treatment, their absence could explain the non-luteolytic effect of PGF(2)alpha injected early in the cycle. Thus, cyclic cows received a PGF(2)alpha analogue and oestradiol valerate together on day 3. This treatment did not affect the life span of the corpus luteum. The absence of oestrogens in the blood does not explain the failure of PGF(2)alpha to cause luteolysis in young corpora lutea.