Determination of oxidative stress parameters and DNA fragmentation on post-thawed buck semen in the presence of ram seminal plasma and fetal calf serum.

The aim of the study was to investigate the efficiency of ram seminal plasma and fetal calf serum on freezing of buck semen. Twenty ejaculates were collected using an electro-ejaculator and split into six groups. While FCS additive was not used in A1, A2 and A3 groups, 10% FCS was added to B1, B2 and B3 groups. These groups were then edited according to whether the buck or ram SP was involved. The design of the groups was done as follows: Group A1 (control 1), group A2 without buck SP, group A3 containing ram SP instead of buck SP. Groups B1 (control 2), B2 and B3 were the FCS added forms of these groups. Progressive sperm motility percentages in Group A1 and Group B2 were found to be higher when compared to the lowest Group B3. There were no significant differences between the groups in neither the levels of reactive oxygen species nor the enzyme and glutathione activities. In conclusion, the lack of statistical difference between the groups suggested that despite the supplements used but only when the buck spermatozoa structure was healthy, the cell could preserve acrosome, DNA and the integrity of membrane.

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes.

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 degrees C while the other was transported at 37 degrees C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 degrees C in a humidified 5% CO(2), 5% O(2), and 90% N(2) atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto-orcein staining. Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI+MII) than the oocytes from anestrual ovaries in the 37 degrees C group (p<0.05). However, oocytes harvested from anestrual ovaries transported at 4 degrees C had the highest maturation (MI+MII) rate, and the difference between anestrual and luteal ovary groups was significant (p<0.05). The oocytes from anestrual ovaries transported at 4 degrees C have significantly higher maturation rates than those transported at 37 degrees C (p<0.0001). However, the transport temperature (37 or 4 degrees C) did not significantly affect the maturation (MI+MII) rates of oocytes harvested from the luteal (p=0.61) and follicular (p=0.48) stage ovaries. It can be concluded from this study that (1) both transport temperature and transport temperaturexestrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 degrees C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.

The potential fertility estimation capacity of the hypoosmotic swelling test, the thermal stress test and a modified cervical mucus penetration test in the bovine.

In this study, hypoosmotic swelling (HOS), thermal stress (TS) and modified cervical mucus penetration (mCMP) tests have been used with routine tests for the assessment of semen quality. This is the first study in which the comparison of potential fertility estimation of fore-mention three tests was performed. Bull semen samples were divided into two fertility groups (high: n=3, low: n=3), according to their post-insemination NRR (non-return rate). Prior to the tests, post-thawed spermatological characteristics were assessed after which HOS, TS and mCMP tests were carried out. In the HOS test, the ratio of swollen cells, in the TS test the motility, and in the mCMP test the number of spermatozoa penetrating the cervical mucus, were examined. The relationship between the tests and fertility was also evaluated. HOS test was carried out according to different incubation times and temperatures (37 degrees C 60 min/41 degrees C 15 min/41 degrees C 30 min/46 degrees C 15 min/46 degrees C 30 min). For TS test, samples were subjected to various temperatures for different periods (no incubation (37 degrees C)/41 degrees C 15 min/41 degrees C 30 min/46 degrees C 15 min/46 degrees C 30 min). The mCMP test were subjected to various temperatures for the same period (37 degrees C 15 min/41 degrees C 15 min). In this study, post-thawed motility was found to be similar in high and low fertility groups. However, it has been determined that acrosomal (p<0.01) and other morphological defects (p<0.05) were low in the high fertility group. When HOS test was carried out at 37 degrees C, no difference was observed between the bulls with high and low fertility, but at 41 and 46 degrees C, results of high fertility group were significantly higher than those of low fertility group (p<0.01). Similarly in TS test, the progressive motility rates of high fertility bulls was higher after thermal practices at 41 and 46 degrees C (p<0.01). In mCMP test, at 37 degrees C, the number of cells that had penetrated was similar. However, significant differences were observed in the incubation at 41 degrees C (p<0.01). It has been concluded that for the estimation of potential fertility of bulls, HOS, TS and mCMP tests, in combination with routine spermatological tests can be used and the use of further penetration distance range (PDR2) in mCMP test and higher temperatures such as 41 degrees C instead of 37 degrees C, during the incubations in the afore-mentioned performance tests, is more determinative.

Relationship between bovine fertility and the number of spermatozoa penetrating the cervical mucus within straws.

In this study, by using a recently developed test technique, the relationship between the total spermatozoa number penetrating determined sites of bovine cervical mucus in straws and potential fertility of bulls, and other spermatological characteristics were investigated. Furthermore, we aimed to determine the effect on the test results, of two different incubation temperatures (37 and 41 degrees C) and two sperm penetration distance ranges (PDRs). Frozen semen samples of six Holstein bulls were used in the study. The bulls were divided into two fertility groups (high and low fertility) according to the "non-return rates" (NRR). For the penetration test, cervical mucus was drawn into transparent plastic straws and incubated with semen at 37 and 41 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, concentrations of spermatozoa penetrated to the PDRs, each of which was 2.5 mm, between 32.5 and 35 mm (first penetration distance range, PDR1), and 50 and 52.5 mm (second penetration distance range, PDR2) distance in the straws from the open end, were measured. When compared with the low fertility group, bulls from the high fertility group showed a higher number of spermatozoa at the determined PDRs, and a significant positive correlation was found between the total number of spermatozoa at the penetration distances and the NRR scores of the bulls.

Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes.

In this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n=23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI+MII. The nuclear maturation rates to MI, MII, and MI+MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p<0.001).

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system.

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), (2)-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription-polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.