The effect of the freezing curve type on bull spermatozoa motility after thawing

The objective of this work was to determine the effect of selected freezing curves on spermatozoa survivability after thawing, defined by its motility. The ejaculates of nine selected sires of the same age, breed, and frequency of collecting, bred under the same breeding conditions including handling, stabling, feeding system and feeding ratio composition, were repeatedly collected and evaluated. Sperm samples of each sire were diluted using only one extender and divided into four parts. Selected four freezing curves – the standard, commercially recommended three-phase curve; a two-phase curve; a slow three-phase curve; and a fast three-phase curve, differing in the course of temperature vs time, were applied. The percentage rate of progressive motile spermatozoa above head was determined immediately after thawing, and after 30, 60, 90, and 120 min of the thermodynamic test (TDT). Moreover, average spermatozoa motility (AMOT) and spermatozoa motility decrease (MODE) throughout the entire TDT were evaluated. Insemination doses frozen using the simpler two-phase curve demonstrated the highest motility values (+2.97% to +10.37%; P < 0.05–0.01) immediately after thawing and during the entire TDT. Concurrently, the highest AMOT (+4.37% to +8.82%; P < 0.01) was determined. The highest spermatozoa motility values were detected after thawing doses frozen by the two-phase freezing curve in eight out of nine sires. Simultaneously, a significant effect of sire individuality was clearly confirmed. Inter-sire differences of spermatozoa motility during TDT as well as AMOT and MODE were significant (P < 0.01). The findings describing both factors of interaction indicate the necessity of individual cryopreservation of the ejaculate to increase its fertilization capability after thawing.

55 EFFECT OF TREHALOSE FOR FROZEN–THAWED RAM SPERM MOTILITY PARAMETERS

Computer-assisted sperm analysers have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Ram semen was collected using electro-ejaculation from 10 adult rams of Chingizskaya indigenous sheep breed. Motility was determined using computer-automated semen analysis (Hamilton Thorne Motility Analyzer, Beverly, MA, USA). Trehalose solution (0.375 M) was added to Tris-buffered saline solution to give the following trehalose extenders: 25, 50, 75, and 100% (vol:vol), and analysed for motility using computer-automated semen analysis. The sperm pellets were resuspended at 24°C in cooling extender – trehalose extenders of each concentration containing 5% egg yolk. The diluted semen was cooled to 5°C within 2 h. The semen was then further diluted 1 : 1 with freezing extender – each trehalose extender containing 1.5% glycerol to obtain a sperm concentration of 2.0 × 108 cells mL–1 – and then loaded into 0.5-mL straws. Straws were frozen using a programmable freezer with a freezing curve of 5°C to –5°C at 4°C per min, –5°C to –110°C at 25°C per min, and –110°C to –140°C at 35°C per min, and then the straws were plunged into liquid nitrogen for storage. Frozen samples were thawed in a 37°C water bath for 30 s and analysed for motility using computer-automated semen analysis. Statistical analyses were performed with a Student's test. The fresh semen samples showed the next results: motility 88.3 ± 2.4%, progressive motility 26.8 ± 6.9%, and progressive velocity 61.9 ± 4.2 μm s–1. Motility of the frozen-thawed spermatozoa was 63.6 ± 2.9% (25% trehalose), 55.6 ± 5.2% (50%), 32.4 ± 4.7% (75%), and 23.6 ± 3.2 (100%). Progressive motility was 15.6 ± 3.9% (25%), 13.7 ± 3.7% (50%), 4.5 ± 1.3% (75%), and 5.2 ± 1.3% (100%). Progressive velocity was 93.5 ± 8.3 μm s–1 (25%), 85.4 ± 8.1 μm s–1 (50%), 65.7 ± 6.1 μm s–1 (75%), 35.2 ± 3.3 μm s–1 (100%). Motility of the frozen-thawed spermatozoa significantly decreased with increasing concentrations of trehalose in the extender (P < 0.05). These preliminary studies showed that further research is needed of use trehalose for ram spermatozoa cryoconservation.

56 COMPARISON OF 4 DIFFERENT DILUENT AGENTS ON CRYOPRESERVATION OF SEMEN FROM UNIMPROVED INDIGENOUS SOUTH AFRICAN GOATS

The cryopreservation process is one of the most crucial factors that affect the post-thaw quality of frozen goat semen. The aim of this study was to compare 4 different semen diluents (Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®). A total of 5 indigenous bucks were used as semen donors. Semen was evaluated for macroscopic and microscopic characteristics. Semen was pooled and diluted randomly in each of the 4 different extenders. Semen samples were equilibrated for 3 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled-rate programmable freezer using a customized freezing curve (from 5 to –5°C at 4°C min–1, –5 to –110°C at 25°C min–1, and then from –110 to –140°C at 35°C min–1). Data was analysed with the aid of the statistical program GenStat®, using a complete randomised design. Following fresh semen dilution with Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, the sperm progressive motility percentage was 41.5 ± 4, 43.5 ± 7, 48.6 ± 5, and 50.2 ± 7%, respectively (P > 0.05). In addition, the live normal sperm percentage following fresh semen dilution was 59.2 ± 5, 49.2 ± 6, 57.8 ± 5, and 54.4 ± 3% for Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, respectively (P > 0.05). After thawing semen samples frozen either with Tris-BSA, Tris-yolk, Bioxcell®, or Ovixcell, the sperm progressive motility percentage was 5.3 ± 2, 1.6 ± 1, 10.6 ± 2, and 13.3 ± 3%, respectively (P < 0.05). Furthermore, the live normal sperm percentage following thawing was 19.1 ± 3, 14.3 ± 3, 39.6 ± 6, and 37.1 ± 3% for Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, respectively. In conclusion, sperm progressive motility percentage was reduced following the freezing-thawing process, irrespective of the type of extender used. The Bioxcell® and Ovixcell® extenders showed to be better for South African indigenous goat semen cryopreservation purposes.

53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P < 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.