Cryopreservation has been reported to damage approximately 40-50% of viable sperm in bull semen. The present study was undertaken to assess the cryo-effectiveness of glycerol (GLY), ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propylene glycol (PND) as cryoprotectant during the cryopreservation of Nguni bull semen. Semen was collected from 18 Nguni bulls and evaluated macroscopically and microscopically for sperm parameters. Thereafter, the semen samples were diluted with egg-yolk citrate extender supplemented with either 12% GLY or DMSO or EG or PND cryoprotectant. Semen samples were loaded into straws and placed into a controlled rate programmable freezer and stored in a liquid nitrogen tank. Following semen thawing, artificial insemination (AI) was done on synchronized Nguni cows. The in vitro fertilization (IVF) was conducted on cow's oocytes to test the fertilizing ability. Data was analyzed with the aid of ANOVA. A significant difference (p < 0.05) was recorded between fresh total sperm motility rate (94.7 ± 2.6%) and frozen-thawed sperm total motility rate with GLY (77.8 ± 11.0%), EG (20.4 ± 10.1%), DMSO (15.7 ± 11.9%) and PND (11.2 ± 11.3%). Interestingly, a positive correlation between total sperm motility and pregnancy rate (r = 0.42) was recorded. However, a negative correlation of Nguni sperm parameters with IVF (r = -0.53) was obtained. The freezing-thawing process did reduce the Nguni sperm total sperm motility percentage.
An improvement in avian semen cryopreservation is essential and has the potential to improve the cryo-gene banking efficiency. This study compared two cryopreservation methods (slow freezing and vitrification) and the effect of different thawing/warming temperatures (5˚C, 25˚C and 41˚C) on Venda cockerel's spermatozoa. Semen samples from Venda cockerels were diluted with modified Kobidil + extender supplemented with 8% dimethyl sulfoxide. Semen from each ejaculate was stained with nigrosin/eosin for viability examination. The cryopreserved samples were either slow cooled in 0.25 mL straw or vitrified in a solid surface vitrification (SSV) device. Semen straw or cryovial was stored in liquid nitrogen container. The straw or cryovial with sperm was thawed or warmed at 5˚C, 25˚C and 41˚C and analysed by a Computer-Aided Sperm Analysis (CASA). There was a significant difference in live/normal sperm between the semen donors. Cockerels spermatozoa cryopreserved by slow freezing (43%) and thawed at 5˚C had a significantly higher survival and motility rate compared to vitrification (2.5%) method. In conclusion, there was higher rate of live/ normal morphology sperm. Cryopreservation process reduces sperm motility and velocity rate regardless of cryoprevervation method and thawing or warming temperatures. However, slow freezing was a better method to maintain motility of spermatozoa following cryopreservation.
The objectives of the current study were to evaluate the estrous response and pregnancy rate following timed artificial insemination (TAI) with frozen-thawed semen in cows. The study was carried out in cows at different villages of KwaZulu-Natal (KZN; n = 160) and Limpopo provinces (L; n = 171). Cows were selected randomly as presented by the farmers, regardless of parity, age, breed and body weight following pregnancy diagnosis. The cows were grouped according to breed type and body condition score (BCS) on a scale of 1-5. Selected cows were inserted a controlled intravaginal drug release (CIDR ®) and removed on day 8, followed by administration of prostaglandin. Heat was observed on day 9 with the aid of heat mount detectors (HMD) that were placed on the individual cow's tail head. Cows on heat were then inseminated twice at 12 hours interval. Pregnancy diagnosis was performed by an ultra-sound scanner and rectal palpation 90 days after TAI. Data were analyzed using SAS 2006. Estrous responses were 100% in KZN and 99% in Limpopo.
Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.
In South Africa, assisted reproductive technologies (ART) such as oestrus synchronization and AI in cattle have traditionally been applied in commercial production systems but not communal production systems because of several challenges such as infrastructure and cost. The study was designed to assess factors affecting response to oestrus synchronization, conception, and calving rate of organised communal cows following timed AI in Limpopo province, South Africa. A total of 140 cows were selected from organised communal villages and categorized according to body condition score (BCS), parity, age, frame size (small to medium) and breed type (Nguni, Bonsmara, and Brahman). A 9-day CIDR® (Pfizer Laboratories, New York, NY, USA) protocol was used to synchronize the selected cows. Heat mount detectors (Karma®; Four Lakes) were used to assess oestrous synchronisation responce. The AI was done twice at 36 and 48 h post-CIDR® removal using Nguni frozen–thawed semen. Pregnancy diagnosis was performed 90 days following AI using ultrasound scanner and trans-rectal palpation. Data on influence of factors such as BCS, parity, age, district, breed type, and frame size on oestrus response, conception and calving rate were analysed using logistic regression procedure of SAS. Of 140 cows synchronized, 75% (105/140) had tripped patches and underwent AI, 41% (43/105) conceived, and 36% (38/105) calved. Parity, age, breed type, and frame size did not significantly affect oestrous synchronisation response, conception, and calving rate. However, BCS significantly (P = 0.0042) affected calving rate, whereby cows in BCS of >3 had a greater probability of success than those with BCS ≤3. Small-framed Nguni and Bonsmara type cows in their first parity with a BCS ≥3 had greater odds of conceiving following timed AI. Noteworthy, calving rate in the current study was comparable to other studies under communal areas (South African Vet. Assoc. 2004 75, 30-36; Appl. Anim. Husb. Rural Develop. 2013 6, 48-54). Therefore, the current study demonstrated an opportunity to improve the production of organised communal cattle using superior sire germplasm though assisted reproductive technologies. Cows in organised communal areas have greater probability of conceiving and calving when their condition score is >3, regardless of their age, parity, size, or breed type. It is concluded, therefore, that AI technology should be applied in cows of organised communal farmers to facilitate dissemination and propagation of superior germplasm.
The invitro embryo production technique is one of the assisted reproduction technologies that has the potential in speeding up genetic improvement in cattle. The developmental competence of invitro-matured oocytes is influenced by several factors during invitro maturation (IVM), such as maturation environment, oocyte quality, type of media, and additives. The objective of the present study was to compare two IVM media (TCM-199 and BO-IVM) on cattle oocyte maturation rate invitro. Cattle ovaries were collected from local slaughterhouse and transported to the laboratory in a thermos flask containing 0.9% saline (Adcock Ingram Critical care) at 37°C. Oocytes were retrieved from the ovaries by the aspiration technique, and then matured invitro in 500µL of TCM-199 (supplemented with 10% fetal bovine serum, FSH, LH, and E2) or in 500µL of commercially available BO- IVM (Bioscience) medium, both covered with mineral oil for 22h at 38.5°C with 5% CO2 and 5% O2. After 22h of IVM, oocyte polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted microscope. The total number of oocytes matured in TCM-199 and BO-IVM media were 401 and 396, respectively. The experiment was replicated 19 times. Data were analysed using the GenStat® program (VSN International). Means of different treatments were separated using Fisher’s protected t-test least significant difference at 5% level of significance. No difference was recorded for polar body extrusion rates in TCM-199 (50.6±13.9) and BO-IVM (47.3±13.7) media. In conclusion, TCM-199 and BO-IVM media did not differ in terms of maturation rate; thus, both can be used for successful cattle oocyte IVM.
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