Asian Pacific Journal of Reproduction

103-116

Authors: P. Perumal

Artificial insemination made a predominant contribution towards the improvement of genetic potential and increased productivity in animal husbandry sectors. In frozen semen technology, about 50% of sperm died because of cryoinjury or cryodamage during the process of cryopreservation and thawing of the semen. These cryo-damages can be minimized by use of various cryoprotective agents or cold shock absorbers in the frozen semen technology. Of which, egg yolk (EY) is most primarily important in the extender preparation for various mammalian species for longer period and it works against the cold shock during the cryopreservation process due to the presence of low density lipoproteins (LDL). Further, EY contains substances other than LDL that affect the sperm quality parameters especially reduced motility and inhibit the respiration of sperm; therefore, there was heavy demand on replacement of the EY with the particular responsible substances (LDL) in the semen extender. Later on, various investigators tried to extract the LDL from the EY of hen and finally succeeded to extract the LDL from the EY of hen for semen preservation of various species. The concentration of LDL used in various species is varied and this may be due to the composition and concentration of phospholipids, cholesterol and its proportion in the sperm membrane. In bovine species, the concentration of LDL was standardized as 8% (w/v) on dry matter basis. This is equal to 20% EY used in conventional semen extender. As it is explained that the 20% EY contains 68% LDL (13.6 g) and on dry matter basis, it is approximately 60% (8.16 g). This calculation indicates 20% EY contains 8% LDL on dry matter basis. The LDL protects the sperm by various mechanisms to maintain the integrity of sperm membrane, which is explained in the present review. It was concluded that the investigation is still to be carried out to find out the exact roles of apoproteins and lipids of LDL and to indentify and isolate the detrimental substances presented in the whole EY.

123-128

Authors: Mojtaba Rashedi, Ali Rooz Batavani, Reza Golam Najafi

Objective: To reveal the effects of 9-cis retinoic acid (RA) on cortical granules (CGs) migration and in vitro embryo production (IVP) rate in meiosis Ⅱ vitrified bovine oocyte. Methods: Following in vitro maturation (IVM) in a medium containing 5 nM of 9-cis-RA, 60 oocytes were vitrified and then thawed. Then, half of them were evaluated for CGs migration and the other half were used for in vitro fertilization and IVP (on day 3). In addition, the other 60 oocytes were considered as the control group, which did not receive RA in IVM medium. The data were presented to one way analysis of variance (ANOVA) and Duncan’s test. Results: Results showed that IVP rate (4-8 cells embryos) was significantly (P<0.05) higher than the control group when RA had been added to IVM medium. Furthermore, the presence of RA in IVM medium improved the rate and mode of CGs migration so that the rate of oocytes that had completed CGs migration in the group, which had received RA, was significantly higher than the control group. Conclusions: This study shows that presence of RA in IVM medium enhances the developmental competence and CGs distribution of meiosis Ⅱ vitrified bovine oocyte. Therefore, adding RA in IVM medium can decrease the ultrastructural changes during vitrification and can improve the efficiency of bovine oocyte vitrification.

136-138

Authors: Umie Lestari, Widodo, Sutiman Bambang Sumitro

Objective: To predict the protein similarity, biological function and structure of 116 kDa protein that was isolated from human sperm membrane of acrosome by using in silico. Methods: Predictions for 116 kDa protein similarity was done through comparative analysis of its isoelectric point (pI) and molecular weight to the UniProtKB/TrEMBL protein database. The three-dimensional structure of the protein was built using SWISS-MODEL. Results: The 116 kDa protein with pI 4.4 was expected to have similarities with CDH23 proteins that had pI 4.38, thus they likely had similar physical and biochemical properties. The predicted protein had an extracellular N-terminal that likely acted as a receptor, and showed cell recognition characteristic, playing a role in fertilization. Conclusions: The 116 kDa protein isolated from acrosome membrane has similarity on its biophysical character to the CDH23.

143-144

Authors: Maryam Eftekhar, Azam Agha-Rahimi, Mohammad Ali Khalili, Marjan Omidi

This paper is a case report on the success of oocyte retrieval and good quality embryo development following controlled ovarian hyperstimulation at early pregnancy. A 30-year-old patient underwent controlled ovarian hyperstimulation by gonadotropin-releasing hormone agonist long protocol. On the day of oocyte collection, a 5-week gestational sac was observed by exact sonography monitoring. However, via ultrasound guided follicle puncture, 7 oocytes were collected. After intarcytoplasmic sperm injection, 3 developed good quality embryos were cryopreserved. Moreover, the natural pregnancy was continued and finally a healthy live birth was achieved. Despite physiological hormonal changes during pregnancy, the follicular growth occurred and followed by oocyte retrieval and embryo development, subsequently.

6-14

Authors: Omolaoye Temidayo S, du Plessis Stefan S

Infertility is prevalent in about 10%-25% of couples in their reproductive age, analogous to 60-80 million infertile couples globally. Of these infertility cases, 10%-30% are exclusively attributed to a problem of the male. Several diseases have been implicated as contributors to deteriorating male fertility and diabetes mellitus (DM) is included. DM, a chronic noncommunicable disease, has been considered as one of the most appreciable health threats, as it affects 9% (422 million) of the world’s population as of 2014. It is characterised by hyperglycaemia, which can result from the inability of the pancreatic β-cells to secrete insulin or from the target tissue becoming insensitive to insulin. DM has been reported to influence male reproductive function through diverse pathways and mechanisms. The adverse effects of reactive oxygen species and successive development of oxidative stress that occur due to DM have been investigated and implicated by several studies. The products of nonenzymatic glycosylation are reported to be widely distributed in the reproductive tract of diabetic men. Additionally, DM has been implicated to impair the processes of male sexual acts. Data reported in this review were extracted from PubMed, Google Scholar, Science Direct and Scopus with diabetes and male infertility as the key search words. In light of the aforementioned, the aim of this review is to provide brief background information on DM as well highlight and explain the likely mechanisms of male fertility which DM impacts

19-21

Authors: Fulvia Gloria-Bottini, Neri A, Pietropolli A, Magrini A, Bottini E

Objective: To investigate the cooperative interaction among five genetic systems (phosphoglucomutase locus 1, adenosine deaminase locus 1, acid phosphatase locus 1, adenylate kinase locus 1, and haptoglobin) concerning their effects on natural fertility in humans. Natural fertility has been evaluated by a model of age related differences between the distributions of types among pregnant women. Methods: A total of 137 nonsmoking consecutive puerperaes from the white population who had delivered their first born baby in the Maternity Department of S. Massimo Hospital of Penne were studied. The phenotypes of the five systems studied were determined by starch gel electrophoresis. Statistical analysis was performed using the statistical package for the social science. Results: There was a highly significant negative correlation between maternal age and the number of genetic factors showing a lower maternal age at the birth of the first child, which suggested a positive cooperative interaction among these factors concerning their effects on fertility. Conclusions: In the relationship of mother-fetus, besides nutritional factors, genetic factors involved in immunological interaction of the embryo with the mother are of paramount importance. Haptoglobin and adenosine deaminase locus 1 polymorphisms are involved in immune reactions and our data indicate that genetic variability within these systems gives a more important contribution to variation of human fertility as compared to acid phosphatase locus 1, phosphoglucomutase locus 1 and adenylate kinase locus 1 that are mainly involved in metabolic functions.

27-32

Authors: Bharath B, Nirmalraj S, Mahendrakumar M, Perinbam K

Objective: To evaluate the effect of marine brown alga Sargassum polycystum extract on growth and biochemical parameters of Vigna radiata and Vigna mungo. Methods: Different concentrations of algal extracts (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, and 5.0%) were prepared and applied to the crops at every 10-day intervals under natural conditions. After 30 d, the plants were harvested to evaluate the growth and biochemical parameters. Results: Seaweed liquid fertilizers treated seedlings showed maximum growth in 3.0% concentration when compared to the untreated seedlings. Similarly, biochemical parameters such as photosynthetic pigments, protein, reducing sugar, total sugar and amino acids exhibited increases in 3.0% concentration seaweed extract. Decreases in growth and biochemical parameters were noticed in concentrations higher than 3.0%. Conclusions: Presence of micronutrients and growth regulating substances in the liquid extract help healthier and faster productivity of the crop.

39-46

Authors: Mohammed KM, Kamal EL-den MA, Dahmoush AY

Objective: To study the genetic and non-genetic factors and their interactions affecting growth rate and body weights at birth, weaning and at 6 months of age in Saudi Ardi, Damascus goats and their crosses. Methods: Crossbreeding program between Saudi Ardi(A) goats with Damascus(D) was carried out to improve the meat productivity of Ardi goats through crossbreeding. The pedigree records of the body weights were obtained from 754 kids (397 males and 357 females) produced from 46 Sires and 279 Dams. Birth weight, weaning weigh and 6 months weight as well as average daily gain during different growth stages from birth to weaning (D1), weaning to 6 months (D2) and from birth to 6 months of age (D3) were recorded during winter/autumn and summer/spring. Data were classified according to breed, generation, sex, season, year, and type of birth. Data were analyzed using GLM procedure for the least-squares means of the fixed factors. Heritability and genetic parameters were estimated with derivative-free restricted maximum likelihood procedures using the MTDFREML program. Results: The percentages of variations were moderate for body weights and high for daily gains. Genetic groups had a highly significant (P<0.01) effect on the body weights traits. Damascus goats had higher (P<0.01) birth and weaning weights, but ½D½A group kids had a higher (P<0.01) body weight at 6 months. The genetic groups had a significant effects on the daily weight gains for D1 (P<0.01) and D3 (P<0.05) periods, whereas, it had no effects on D2 period. The fixed effects of sex, season, year and type of birth were significant differences for body weights. Male kids were heavier (P<0.01) than females for different growth stages. Body weights and daily gains during winter/autumn were significantly higher (P<0.01) than summer/ spring. Kids born and raised as singles were significantly (P<0.01) heavier than those were born as twins or triplets. The genetic and phenotypic correlations between birth and weaning weights were positive for both Damascus and Ardi goats. Conclusions: Genetic program for Ardi goats through upgrading with Damascus is possible to improve meat production.

247-251

Authors: Nahid Yari, Sahabeh Etebary, Mohammad Ali Khalili, Ali Reza Talebi

Objective: To evaluate the impact of freezing–thawing on the human sperm head vacuoles and the potential value of motile sperm organelle morphology examination for selection of frozen-thaw spermatozoa. Methods: In 30 sperm samples from infertile men, analysis for conventional sperm parameters (motility, vitality, and normal morphology) and a morphological analysis at high magnification for vacuoles examination were done before cooling and after warming. For description of sperm head vacuoles, two hundred spermatozoa were examined and were classified into three groups according to presence and vacuole areas including no vacuole group (free of any vacuole), small vacuole group (occupy not more than 4% of the nuclear area), and large vacuole group (occupy more than 4% of the normal nuclear area). Results: Significant reduction of progressive motility and vitality was observed following cryopreservation (P<0.001). Also, normal morphology decreased significantly after cryopreservation (P<0.05). Spermatozoa with a vacuole free head had a significant reduction in cryopreservation group (P=0.013). The percentage of spermatozoa with small vacuoles increased slightly, but not significantly after cryopreservation (P=0.296). Conclusions: Motile sperm organelle morphology examination is a powerful research tool for investigating spermatozoa abnormalities such as vacuoles that are increased post cryopreservation.

257-263

Authors: Asmarinah, Tri Panjiasih Susmiarsih, Amalia Shari, Putri Ratri, Dwi Ari Pujianto, Endang Winiati Bachtiar

Objective: To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded. Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly. Conclusions: Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

268-278

Authors: Perumal P, Savino N, Sangma CTR, Khan MH, Ezung E, Chang S, Sangtam TZT

Objective: To analyse the seasonal effect on physiological parameters, reproductive profiles and in vitro fertility in breeding mithun bulls. Methods: A total of ten adult mithun bulls age of 5 to 6 years old with good body condition (score 5-6) were selected from ICAR-NRC on Mithun, Jharnapani, Nagaland, India. The seasons were categorised into winter, spring, summer and autumn seasons based on the meteorological data and sunshine hours. The physiological parameters, reproductive profiles and in vitro fertility parameters were assessed during different seasons in mithun under the semi-intensive system of management. Results: The statistical analysis revealed that these experimental parameters were differed significantly (P<0.05) among the seasons and in overall spring and winter seasons were more beneficial in mithun breeding programme, although, the breeding in mithun occurred throughout the year with variation. Conclusions: It is concluded that collection & preservation of mithun semen and artificial insemination in mithun species during the season of spring and winter has significant beneficial effect in terms of semen production, freezability and fertility for artificial breeding programme in mithun under the semi-intensive system.

283-288

Authors: K.H. El-Shahat, M. I. Taysser, M.R. Badr, K.A. Zaki

Objective: To determine the effects of different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) as well as incubation time on motility, hyperactivity and acrosome reaction (AR) of ram sperm in vitro. Methods: Freshly ejaculated spermatozoa from three ram were collected, pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate medium supplemented with different concentrations of PHE (10, 20, 30, 40, 50, 75 and 100 mM/mL). Then best concentrations were compared and examined for motility, hyperactivity and AR. Results: A high concentrations of PHE (30, 40, 50, 75 and 100mM/mL) showed a significant increase in motility when compared to control immediately after dilution and exist for the first and second hour of incubation period. However, when longer incubation time were used, a significant (P<0.05) decrease in motility was achieved. Similar finding was observed in hyperactivity. Stained semen samples showed a maximum percentage of incomplete AR after 1 h incubation corresponding to 50 and 75 mM/mL of PHE; however, spermatozoa treated with 75 mM/mL had a higher tendency to undergo complete AR after further incubation up to 4 h. A dose dependent relationship was detected where the maximum value of total AR was shown in spermatozoa treated with 75 mM/mL PHE for 4 h. Conclusions: To obtain better motility, hyperactivity and AR, treatment of ram spermatozoa with 75 mM/mL PHE for 4 h before being used in insemination was considered the best concentration of PHE to be used in the process of in-vitro fertilization.

197-201

Authors: Hanieh Vaseghi, Asghar Mogheiseh, Mojtaba Kafi, Masood Sepehrimanesh, Mohammad Hossein Nooranizadeh

Objective: To study the effects of anestrus dog serum in rodents (contains high amount of FSH) compared with two common superovulatory programs. Methods: Rats (n=30) at diestrus phase were evenly divided into pregnant mare serum gonadotrophins (PMSG) group (administrated with 30 IU PMSG, and 48 h later following by 25 IU hCG), recombinant follicle stimulating hormone (rFSH) group (reducing dose every 12 h from 5 to 1 IU, then following by 25 IU hCG) and anestrus dog serum group (reducing dose every 12 h from 0.6 to 0.1 mL, then following by 25 IU of hCG). Mice (n=30) were also evenly divided into PMSG group (5 IU PMSG used at 13 pm, and 48 h later following by 5 IU of hCG), rFSH group (from 13 pm, reducing dose every 12 h from 2.5 to 0.5 IU, then following by 5 IU hCG) and anestrous dog serum group (from 13 pm, reducing dose every 12 h from 0.100 to 0.025 mL, then following by 5 IU hCG). Mice and rats placed with males for 24 h after last injection. Histology samples of ovaries were prepared and the numbers of corpus lutea were counted on day 14 after mating. Results: In mice, the differences among mean number of corpus lutea in all groups (P=0.01) and between FSH and dog serum group were significant (P=0.000 7). But no significant differences were found between the mean number of corpus lutea in dog serum and PMSG, or between FSH and PMSG groups. In rats, mean number of corpus lutea were significant differences among three groups (P=0.01), and between PMSG and dog serum groups (P=0.02). Conclusions: Superovulatory response in anestrus dog serum group is similar to PMSG group in mice, which is relatively similar to FSH in rats.

212-216

Authors: Irfan Yunianto, Nurul Ain Kamar Bashah, Mahanem Mat Noor

Objective: To investigate the antifertility properties of Centella asiatica L. ethanolic extract and identify the sperm proteomic changes in treated male rats. Methods: A total of 32 male Sprague-Dawley rats were divided into two groups: control group (n=16) received distilled water and treatment group (n=16) which received 300 mg/kg of Centella asiatica L. extract for 42 d. After the treatment period, the number of implantation sites was recorded and the sperm proteomic changes were analysed by 2D gel electrophoresis. In addition, the expression of protein spots was quantified by MALDI-TOF analysis. Results: Centella asiatica L. extract resulted in low number of implantation sites in the treatment group (100.00±2.82) compared to the control group (183.00±2.14). The percentage of infertile male rats in the treatment group was higher (43.75%) compared to the control group (18.75%). Proteomic analysis showed the expression of protein spots identified in the treatment group decreased with 234 spots compared to the control group with 282 spots. Conclusions: The results revealed that there was antifertility activity in the male rats with the administration of Centella asiatica L. ethanol extract and the identified proteins could provide understanding on the adverse effect of Centella asiatica in male reproductive system.

221-225

Authors: Fabio Diniz de Souza

Objective: To explore some possibilities of computer applications in medicine, and to discuss an online infertility clinical decision support system. Methods: Retrospective data were obtained from 52 couples, and then entered into the online tool. Both its results and the initial diagnoses obtained by the treating physicians were compared with the final diagnoses established by laparoscopy and other diagnostic tests (semen analysis, hormone analysis, endometrial biopsy, ultrasound and hysteroscopy). The initial hypothesis of the research was that the online tool’s output was statistically associated with the final diagnoses. In order to verify that hypothesis, a chi-square (氈2) test with Yates’ correction for continuity (P<0.05) was performed to verify if the online tool’s and the doctor’s diagnoses were statistically associated with the final diagnoses. Results: Four etiological factors were present in more than 50% of the couples (ovarian, tubal-peritoneal, uterine, and endometriosis). The statistical results confirmed the research hypothesis for eight out of the nine etiological factors (ovarian, tubal-peritoneal, uterine, cervical, male, vaginal, psychosomatic, and endometriosis; P<0.05). Since there were no cases related to the immune factor in the sample, further clinical data are necessary in order to assess the online tool’s performance for that factor. Conclusions: The online tool tends to present more false-positives than false negatives, whereas the expert physician tends to present more false-negatives than false-positives. Therefore, the online tool and the doctor seem to complement each other. Finally, the obtained results suggest that the infertility online tool discussed herein might be a useful research and instructional tool.

231-237

Authors: Mohamed El-Raey, Heba M El-Khaiat

Objective: To assess the anion gap (AG) validity in prediction the goat’s fertility before parturition. Methods: This study was carried out on two groups (20/20, aged 4–6 years and weighted 30–43 kg) of pluriparous pregnant Baladi goats. Animals were subjected to clinical examination, blood sampling to assess serum biochemical parameters in correlation with utrasonographic assessment of uterine horn involution (UTI) and placental dropping time after kidding. Unpaired student t–test was used to detect the significance of differences (P<0.05). Correlation coefficient was used to study the relationships among the different assessed parameters, analysis (r) was assigned at P<0.05 using the same statistics program. Results: Results revealed that there were highly significant differences (P<0.05) between normally and abnormally parturated goats concerning Cl–, K+, Na+, P, Mg2+, Total/Ca2+, albumin, glucose and AG. Goats suffered from dystocia having high levels of HCO3 –, cortisol and lactate. Additionally, there were significant differences (P<0.05) between normally and abnormally parturated goat does regarding the process of UTI starting from 3rd to 10th wk after parturition. Furthermore, there was a highly significant difference (P<0.05) between normally and abnormally parturated goat does concerning their placental dropping time; where, distressed/abnormally parturated goat having the longest time. Furthermore, there were highly strong correlation coefficient between Mg2+, K+, Na+, HCO3–, glucose, albumin, AG and corrected AG concentrations during goat’s prepartum period and UTI, placental dropping time. Conclusions: Reporting serum bio-chemical changes and AG during goat’s prepartum period could be used as diagnostic tool to judge the normality of parturition process, placental dropping, UTI and genital health, in other word, could be used as predictor for goat’s fertility prior to parturition.

151-157

Authors: Deotima Sarkar, Arijit Chakraborty, Dakshayani Mahapatra, Amar K Chandra

Objective: To evaluate the effect of daily consumption of bamboo shoots (BS) on the morphological features and functional status of the female reproductive system in adult with respect to thyroid. Methods: Adult female rats were divided into control and experimental groups of six each. Control group was given normal diet while experimental group was fed BS by 1/3rd replacement of 180 g of their food i.e. 60 g of BS containing 35 g of goitrogens of cyanogenic origin such that each rat likely consumed 6 mg/100 g of body weight per day for a period of 45 d. Morphological features like changes in body weight and organ weight were noted. Key steroidogenic enzyme levels viz Δ5 3β hydroxysteroid dehydrogenase (HSD) and 17β HSD along with serum estradiol, estriol and progesterone levels were measured. Estrous cyclicity of the animals monitored regularly followed by histological analysis of thyroid, ovary and uterus at the end of experimentation. Results: Increase in body weight, thyroid gland weight and thyroid stimulating hormone, decrease in serum triiodothyronine and thyroxine, a decrease in ovarian as well as uterine weight and the activity of steroidogenic enzymes Δ5 3 β HSD and 17β HSD along with diminished serum estradiol, estriol and progesterone levels were noted; while histological plates showed prominent degenerative changes in both the ovary and uterus. Estrous cyclicity of the treated animals were irregular and almost stopped at diestrous stage of the cycle in the latter stage of the treatment as compared to control. Conclusions: Overall results indicates that BS rich in cyanogenic constituents induces biochemical hypothyroidism in the experimental animals that acts in corroboration to cause morphological and functional alteration of reproductive organs indicating its likely impact in fertility on continued use.

164-171

Authors: Maryam Owjfard, Manzarbanoo Shojaei Fard

Objective: To evaluate the effect of long-term exposure to radiofrequency radiations emitted from mobile jammers onrat's reproductive parameters. Methods: Eighty male rats were divided into neonatal group (n=20), immature group (n=30) and mature group (n=30). Neonatal rats were randomly divided into two subgroups. Immature and mature rats were randomly divided into three subgroups. Experimental rats were fixed in the restraining tube and were exposed to radiofrequency radiations emitted from mobile jammers at a distance of 100 cm, for 30 d (7 h/d). Sham group rats were also fixed in restraining tube but without radiations. The control group was allowed to move freely without being exposed to radiation. At the end of the experiment, weight of testes, sperm quality, histological testicular sections and serum testosterone levels were evaluated. Results: Long-term exposure to radiofrequency from mobile jammers could adversely affect neonatal rat fertility, but it did not have significant effects on male mature and immature rat's reproduction parameters. However, restraint stress induced by immobilizing them for a long-period could adversely affect male mature rat's reproductive parameters, but it had no significant effect on male immature rat's reproduction parameters. Conclusion: The effects of EMF exposure to be various based on the life stage.

176-180

Authors: Mohamed MM El-Sokary, Mohamed El-Raey, Karima GhM Mahmoud, Mahmoud EA Abou El-Roos, Gamal MS Sosa

Objective: To assess the effects of melatonin and/or cysteamineon in vitro maturation, culturing and post-warming of buffalo embryos. Methods: Buffalo oocytes were classified into control, cysteamine (50 μM), melatonin (10 ng/mL) and cysteamine (50 μM) + melatonin (10 ng/mL) treatment groups. In experiment 1, previous treatments were added during in vitro maturation and culturing of buffalo oocytes. Results: Cleavage and blastocyst rates were significantly (P<0.05) increased in melatonin treated group (70.5±0.9 and 12.8±1.0, respectively). However this effect was potentiated when combined with cysteamine (74.0±1.7 and 14.8±1.7, respectively). In experiment 2, the treatements were added in maturtaion, culturing as well as post-warming culture media. Embryos at 7 d were vitrified.Viability assessement directly after warming showed significant increase (P<0.05) in cysteamine, melatonin and their combination groups (76.8±2.8, 80.0±2.1 and 83.3±1.7, respectively) than control (65.8±2.4); but the viability after 24 h post-warming was the best in cysteamine + melatonin combination group (61.4±2.1). Conculsions: Enriching maturation, culturing and post-warming media of buffalo oocytes and embryos with melatonin and/or cysteamine have significantly beneficial effects on oocyte developmental competence as well as embryos vitrification procedure outcomes which in turn resulting in enhancement of commercial buffalo embryo production.

186-190

Authors: Kanoknan Somnuk, Pim Wannapakorn, Waraporn Raksapol, Bunlue Kornmatitsuk, Sudsaijai Kornmatitsuk

Objective: To establish fetal age equations based on ultrasound measurements in cross-bred Holstein cows in Thailand. Methods: The animals were bred for 30-120 d before pregnancy diagnosis using transrectal ultrasonography with real-time B-mode and specify 5 or 7.5 MHz linear array transducer. Parameters examined included crown rump length (CRL), trunk diameter (TrD) and eye diameter (ED). Results: One hundred and two cows had been examined, 80 of them were pregnant and contained at least one measurable characteristic as defined in the methods. Regression analysis and curve estimation were implied. The most frequently visualized parameters were TrD (42/80; 52.50%) and CRL (41/80; 51.25%) whereas ED (13/80; 16.25%) was least frequently detected. In our study, the equations to estimate fetal age (indicated as Y) from CRL and TrD were established with very high correlation coefficients as follows, Y = 22.679+12.005 (CRL)-1.042 (CRL)2, R2 = 0.950, P<0.001 and Y = 14.583+29.878 (TrD)-3.759 (TrD)2-0.225 (TrD)3, R2 = 0.950, P<0.001. The equation from ED was Y=107.582-98.928 (ED)+61.116 (ED)2-9.221 (ED)3 but with very low correlation coefficient (R2 = 0.673, P=0.021). Comparing between our equations (CRL and TrD) and the ones embedded in the commercially available ultrasounds, the higher fetal age was estimated based on the same value of parameters (P<0.05). In conclusion, the most reliable equations created in our study were from CRL and TrD. Conclusions: Comparing with others, Thai cross-bred fetus clearly showed slower growth rate and thus, to use our CRL’s and TrD’s equations for estimating fetal age seems more reliable than to use the ones embedded in the commercial ultrasounds.