Rafael Herrera Alvarez

Transfer of Bovine Blastocysts Derived from Short-term In Vitro Culture of Low Quality Morulae Produced In Vivo

The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus or TCM-199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5 degrees C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non-cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor-recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non-cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non-cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short-term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.

The use of lower doses of the prostaglandin analogue, cloprostenol, for oestrus synchronization in heifers

Abstract In an attempt to reduce costs for cattle oestrus synchronization, the use of lower doses (50 and 25% of the conventional dose) of the prostaglandin F2α analogue, cloprostenol, given by either intramuscular (IM) or intravulvo-submucosal (IVSM) injection, was investigated. There were no differences in the luteolytic response, as measured by either the fall in plasma progesterone or onset of oestrus, between lower cloprostenol doses administered by either method and 500 μg given by IM injection.

Superovulatory Response of Zebu Cows Treated with pFSH in a Single Subcutaneous Injection Followed by an Additional Intramuscular Sub-Dose 48 h Later

The aim of this study was to evaluate whether an additional intramuscular (im) injection of pFSH would increase the embryo production in zebu cows superovulated with a single subcutaneous (sc) pFSH injection. Twenty-one Nelore cows were treated with a progesterone vaginal implant (Controlled Internal Drug Relased - CIDR B) and injected im with 2.5 mg estradiol benzoate. Four days later, cows were assigned randomly into three groups and superovulated with pFSH. Groups A and B received single sc injections of 400 and 320 IU, respectively; Group C received multiple im injections of 400 IU in decreasing doses at 12-h intervals over 4 days. In the morning (07:00 am) of day 3 after starting superovulation, cows received im 150 mcg cloprostenol and Group B was additionally injected im with 80 IU of pFSH. CIDR-B was withdrawn in the afternoon (07:00 pm). Cows were inseminated 48 and 62 h after the cloprostenol injection. Embryo collection and corpora lutea (CL) estimation were done 7 days after insemination. Alternation of treatments (crossover design) occurred at a 60-day interval. There was no significant difference (p > 0.05) of CL counts among treatments. The total (transferable and no transferable) number of recovered embryos from Group A (6.9 +/- 1.5) was not different from Group C (9.8 +/- 1.2), whereas Group B (5.7 +/- 1.5) was lower than Group C (p < 0.05). The number of transferable embryos from Groups A (2.4 +/- 0.7) and B (1.7 +/- 0.6) was lower (p < 0.05) than Group C (4.6 +/- 1.2). Lesser (p < 0.05) embryo production from Group B was related to lower recovery rate (46.4%), compared with Groups A (65.1%) and C (81.7%). It was concluded that an additional im subdose of pFSH, injected 48 h after a single subcutaneous (sc) dose of pFSH, does not improve the embryo production in zebu cows.

Eficácia do tratamento Ovsynch associado à inseminação artificial prefixada em rebanhos Bos taurus e Bos indicus

The present study aimed to evaluate the efficiency of Ovsynch treatment associated with prefixed-time artificial insemination on Bos taurus and Bos indicus cows. Herds of Holstein, Caracu, Nelore and Mantiqueira breeds were used. A herd of Gir cows with poor reproductive performance was also included. Each breed was divided in tree groups. Group 1 received Ovsynch treatment and was inseminated at prefixed time. Group 2 was induced to estrus with cloprostenol and inseminated thereafter. Group 3 was inseminated at natural heat. Conception and pregnancy rates were determined by ultra-sound. No significant interactions were detected among breeds, age, parity, postpartum period and lactating cows with or without their calves. Conception rates did not differ (P>0.05) among groups while pregnancy rate was higher (P<0.05) for group 1 and 2 comparing to group 3. In the Gir herd Ovsynch treatment did not enhance conception nor pregnancy rates. Independently of breed, conception rate is similar after Ovsynch, cloprostenol and control treatments, and pregnancy rate can be improved with Ovsynch and cloprostenol treatments. In cows with unspecific reproductive ovarian disorders, Ovsynch treatment do not enhance conception or pregnancy rates.

Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin

Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health Organization. The peaks obtained by this qualitative and quantitative physical–chemical analysis were compared using an in vivo bioassay based on the ovarian weight gain of prepubertal female rats. The RP-HPLC data showed one or two peaks close to a main peak (tR = 27.9 min), which were related to the in vivo bioactivity. Commercial preparations that have this altered peak showed very little or no in vivo activity, as demonstrated by rat ovarian weight and in peripubertal gilts induced to ovulate. Overall, these findings indicate that RP-HPLC can be a rapid and reliable tool to reveal changes in the physicochemical profile of commercial eCG that is apparently related to decreased biological activity of this hormone.

Produção in vitro de embriões bovinos: utilização de diferentes fontes de gonadotrofinas na maturação dos oócitos

This study aimed to evaluate the effect of different gonadotropin sources for in vitro maturation of bovine oocytes fertilized and developed in vitro on cleavage (CR) and blastocysts rates (BLR). The immature oocytes from slaughterhouse cows ovaries were matured in vitro under different conditions: TCM 199 medium with 10% eostrous cow serum (ECS), aditives, hepes, NaHCO3, sodium pyruvate, antibiotics (B-199 medium), 20 UI/mL PMSG and 10 UI/ml hCG (PMSG/hCG) or B-199 medium with 5 mg/mL FSH and 5 mg/mL LH (FSH/LH). After 24 hours of culture at 38.5°C with 5% CO2 in air the mature oocytes were incubated with frozen-thawed semen for 18-21 hours. After this, the oocytes were transferred into microdroplets of Menezo medium with 10% ECS and bovine oviduct ephitelial cells (BOEC) in suspension and were further cultured for 9 days. The data were analyzed by chi-square test. The CR and BLR for PMSG/hCG and FSH/LH were 60 and 13.9% and 61.2 and 10.6%, respectively. There was no difference between treatments regarding to CR and BLR. The results indicate that both gonadotropin sources can be used for in maturation medium of oocyte fertilized and developed in vitro.

Endocrine profiles and ovulation rate of cows superovulated with FSH following passive immunization against steroid free-bovine follicular fluid

n spite of the considerable progress in embryo splitting and in vitro fertilization techniques, superovulation with gonadotrophins is still the most feasible and widely used process for both research and applied embryo transfer programs in cattle13. The major disadvantage of gonadotrophin treatment is the great variability of ovarian response between and within animals3,5,12. For instance, data analysis from 960 cows treated for superovulation during a 10-year period (in the Embryo Transfer Unit of the Instituto de Zootecnia, Nova Odessa, SP, Brazil) showed an average of 6.1 recovered embryos but the standard deviation was 5.1. The two most important factors affecting variability in ovarian response are: a) gonadotrophin treatment per se3,19 and b) ovarian status at the time of gonadotrophin treatment17,18. There have been many attempts to improve the superovulatory response. Using different gonadotrophin preparations or increasing the number of follicles available for stimulation by pretreatment with FSH or GH have been somewhat successful in increasing the ovulation rate and the number of transferable embryos. However, these methods did not reduce the variability of ovarian response. Initiating superovulation in the absence of a functionally dominant follicle is an efficient way of increasing the number of recovered embryos and reducing the variability. The disadvantage of such a method is that it requires either an ultrasonic monitoring or a surgical electrocautery. The dominance of growing follicles can be suppressed using immunological techniques as shown in non-superovulated cattle passively immunized against inhibin or charcoal-treated bovine follicular fluid. The use of inhibin for immunization is somewhat limited by cost and availability. In contrast, bovine follicular fluid can be readily aspirated from abattoir-derived ovaries. In the studies above, the ovulation rate was moderately increased by immunization. Therefore, it is possible that such a method can also contribute to render the animals more sensitive to exogenous gonadotrophins. Endocrine profiles and ovulation rate of cow superovulated with FSH following passive immunization against steroid free-bovine follicular fluid*

Efeito do líquido folicular bovino tratado com carvão ativado na secreção de FSH em novilhas pré-púberes intatas e ovariectomizadas

The aim of the experiment was to determine whether charcoal-treated bovine follicular fluid (bFF) removed from visible (< 22 mm) follicles altered the secretion of follicle-stimulating hormone (FSH) in intact and ovariectomized prepubertal heifers. After a 10-ml injection of bFF given at three consecutive 8-h intervals, secretion of FSH was depressed in approximately 44% of the ovariectomized heifers but there was no effect in intact heifers. When the bFF treatment ceased, there were no rebound effects on FSH concentrations above that of controls pretreatment levels. These results suggest that proteins from bFF act at the pituitary level to inhibit FSH secretion and, differently of the intact, the ovariectomized heifer is an adequate model to put this effect in evidence, particularly when the bFF have low FSH suppressing activity.