Nuclear maturation of immature bovine oocytes after vitrification using open pulled straw and cryotop methods

To date, at least two well known methods have been widely used for vitrification of oocytes and embryos at different stages in a variety of species. However, there is no reported data regarding the comparative effectiveness of these two methods for vitrification of immature bovine oocytes. The objective of this study is to compare the nuclear maturation of immature bovine oocytes vitrified using open pulled straw (OPS) and cryotop methods. Two experiments were conducted in this study. In the first experiment, cytotoxicity of vitrification solutions (VS) from both methods was studied. After removal of cryoprotectants, cumulus oocyte complexes (COCs) was cultured in vitro and cleavage rate was monitored on Day 2 post-insemination (pi), whereas, morulae and blastocyst yields on Days 5 and 8 pi, respectively. The VS solutions significantly reduced zygotic cleavage rate, morulae and blastocyst rates compared with the control group (P < 0.05). The lowest cleavage rate resulted from prolonged exposure time to OPS-VS solutions (35.1%; P < 0.05). However, the morulae and blastocyst rates were significantly higher (P < 0.05) for embryos derived from oocytes exposed to cryotop solutions (40.5 and 22.4%, respectively). In the second experiment, effectiveness of both vitrification methods was compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop solution and device showed higher percentages of PB+ (36%) and MII (51%) rates. In addition, the lowest percentage of degenerated oocytes resulted from cryotop solution. The highest degenerated oocytes obtained by equilibration in OPS solution and vitrified using OPS device (40%; P < 0.05). In conclusion, our data demonstrated that cryotop solution was less toxic to the immature bovine oocytes and vitrification with the cryotop method resulted in higher survival and nuclear maturation rates.Key words: Immature oocyte, bovine, vitrification, cryotop, open pulled straw (OPS)

Morphometric and Histological Characteristics of the Uterus of Rusa Deer (Rusa timorensis) during Oestrus

The purpose of this study was to provide baseline data on the histological characteristics of the uterus of an uncommon tropical deer species, Rusa deer (Rusa timorensis) in captivity. Samples (1cm3) of the the entire parts of the uterus (horn, body and neck) from seven (n = 7) Rusa timorensis hinds in oestrus (exhibiting receptivity to the stag the day before slaughter) were obtained from Universiti Putra Malaysia deer breeding unit and fixed in 10% formalin and 4% gluteraldehyde for light and scanning electron microscopy respectively. In the utero-tubal junction, simple tubular glands, similar to endometrial glands were observed. In the endometrium, ciliated cells were scattered among the secretory cells with proliferation of straight endometrial uterine glands which are lined by tall columnar cells with plasma cells at the basement membrane. These glands were extremely well developed and their histological structure indicates increased secretion preparatory to pregnancy. The study demonstrated that the Rusa deer showed increased reproductive parameters as well as parameters manifested in venison production. The microstructure of uterus of Rusa deer shows extremely positive characteristics for nidation of the egg cell and normal development of the embryo. From these investigations, it is clear that the microstructure of the uterus shows favourable conditions that allow for proper development of a healthy embryo even in captivity.Key words: Captivity, histology, oestrus, Rusa timorensis, uterus

Anatomy of the female reproductive system of Rusa deer (Rusa timorensis)

The study aims to present baseline data on the reproductive anatomy of a poorly known tropical deer  species, Rusa deer (Rusa timorensis). The anatomy of female reproductive system is described using  seven uniparous hinds, aged between four and eight years. The various reproductive structures were  studied via standard descriptive methods. There was a significant difference in the length and width of both  right and left ovaries. The left ovary was slightly larger than the right ovary which indicates that it is physiologically more active. The results of the study showed that the anatomy of female reproductive  system of R. timorensis was similar to that observed in domestic ruminants except that the uterus did not  have an interconual ligament and this implies that the uterine horns are anchored in such a way that sperm deposited into only one uterine horn of the Rusa deer will be transported to the other uterine horn  (interconual transport). Unlike the red brocket deer and pampas deer, the cervix of R. timorensis was  characterized by six cervical rings projecting into the cervical canal. This feature should be taken into  account when designing effective instrumentation and techniques for transcervical passage of semen during  artificial insemination in this species. The results from this study have provided baseline data on the reproductive anatomy of this vulnerable species, and the knowledge generated can be useful in the  development of appropriate reproductive techniques in order to increase its population in captivity and also enable easy detection of its reproductive anomalies, thus strategies to propagate and conserve the species can be established.Keywords: Anatomy, Female, Reproductive system, Rusa deer, Timorensi

Fecal Progestin Extraction and Analysis for Non-invasive Monitoring of Ovarian Cycle in Beef Cows

The aims of the present study were to determine presence of immunoreactive progestins in feces, correlate fecal progestins with plasma progesterone (P4) concentrations and subsequently assess the role of fecal progestins in monitoring estrous cycle in Kedah Kelantan (KK) beef cows. A total of 12 cycling cows were subjected to blood and matched fecal sampling twice a week for 9 weeks. The concentrations of plasma P4 and fecal progestins extracted using a modified technique, were determined by a P4 radioimmunoassay (RIA) kit. There was a significant positive correlation between the concentrations of fecal progestins and plasma P4 (r = 0.6, P<0.01), as tested for the whole group except one animal. High performance liquid chromatographic separation of fecal extracts and subsequent radioimmunoassay revealed presence of four immunoreactive progestins against the P4 antibodies. These results imply that the non-invasive measure of fecal progestins using a DSL-3900 RIA kit can be used to monitor the ovarian activity in beef cows

Identification of bovine growth hormone (BGH) gene polymorphism using PCR-RFLP method in buffalo bulls

Growth Hormone (GH) is a single polypeptide chain synthesised and secreted from anterior pituitary gland by somatroph cells. The product of GH gene hastens metabolism and promotes the growth of many organs and tissues especially bone, muscle and visceral organs. It also regulates growth, mammary gland development and lactation. Polymorphism in this gene is associated with increase in growth and development of many tissues in the body. Aim: The objective of this study was to investigate the polymorphism of bovine growth hormone (bGH) gene in buffalo bulls (Bubalus bubalis) using the PCR-RFLP (polymerase chain reaction–restriction fragment length polymorphism) technique. Design: Genomic DNA was extracted from a total of 10 bulls, consisting of Murrah – Swamp crossbred and pure Swamp buffalo bulls. A The 446 segment of the bGH gene was amplified. The DNA amplicons were detected in 2% agarose gel following 45 minutes of electrophoresis. They were thereafter digesting with AluI endonuclease restriction enzyme, and the digested DNA were detected in 2% agarose gel following electrophoresis for about 45minutes in all samples Results: Similar bands of approximately 300 and 146-bp each, with no variation, were detected in 2% agarose gel following electrophoresis in all the animals tested. Conclusion: Based on the Alu1 digestion result, all samples produced the same allele of the gene, with no polymorphism detected

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen

Aim: The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters. Materials and Methods: A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test. Results: The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups. Conclusion: In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment

Effects of timed artificial insemination following estrus synchronization in postpartum beef cattle

The objectives of this study were to evaluate estrus response and pregnancy rates resulting from timed  artificial insemination (AI) following estrus synchronization using CIDR in postpartum beef cattle. A total of  100 cows were randomly divided into three groups. Groups 1, 2 and 3 were artificially inseminated at 48-50 h (n=30), 53-55 h (n=30) and 58-60 h (n=40) after CIDR removal, respectively. Estrus synchronization was  carried out using a CIDR containing 1.38 mg progesterone. All cows were given 2 mg estradiol benzoate,  intramuscularly on the day of CIDR insertion (D 0). The CIDR was removed after 8 days and 125 µg of  prostaglandin F2á (PGF2α) was injected intramuscularly. One day after CIDR removal all cows were given 1  mg of estradiol benzoate intramuscularly (D 9). Cows were observed visually for estrus after removal of CIDR.  Between 30 and 32 days after timed AI, pregnancy was determined using transrectal ultrasonography. The first estrus observation which is approximately 32 h after CIDR removal showed no significant difference  (P&gt;0.05) among the three groups. The onset response of estrus after 32 h removal of CIDR was less than 10% in all three groups 6.6% (G1), 6.8% (G2) and 7.3% (G3). Furthermore, percentages of estrus response (D 10)  following CIDR removal were 76.6%, 75.0% and 77.5%. The difference between on D 9 and D 10 estrus  response were statistically significant (P&lt;0.05). The pregnancy rates were 23.3% (G1), 26.6% (G2) and 37.5% (G3), which were not significant (P&gt;0.05).Keyword: Cows, Estrus synchronization, CIDR, Timed artificial insemination (TAI), Pregnancy rat

Effects of timed artificial insemination following estrus synchronization in postpartum beef cattle

The objectives of this study were to evaluate estrus response and pregnancy rates resulting from timed artificial insemination (AI) following estrus synchronization using CIDR in postpartum beef cattle. A total of 100 cows were randomly divided into three groups. Groups 1, 2 and 3 were artificially inseminated at 48-50 h (n=30), 53-55 h (n=30) and 58-60 h (n=40) after CIDR removal, respectively. Estrus synchronization was carried out using a CIDR containing 1.38 mg progesterone. All cows were given 2 mg estradiol benzoate, intramuscularly on the day of CIDR insertion (D 0). The CIDR was removed after 8 days and 125 μg of prostaglandin F2α (PGF2α) was injected intramuscularly. One day after CIDR removal all cows were given 1 mg of estradiol benzoate intramuscularly (D 9). Cows were observed visually for estrus after removal of CIDR. Between 30 and 32 days after timed AI, pregnancy was determined using transrectal ultrasonography. The first estrus observation which is approximately 32 h after CIDR removal showed no significant difference (P>0.05) among the three groups. The onset response of estrus after 32 h removal of CIDR was less than 10% in all three groups 6.6% (G1), 6.8% (G2) and 7.3% (G3). Furthermore, percentages of estrus response (D 10) following CIDR removal were 76.6%, 75.0% and 77.5%. The difference between on D 9 and D 10 estrus response were statistically significant (P0.05)