Influence of seasonal differences on semen quality and subsequent embryo development of Belgian Blue bulls.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14-28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group (P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group (P < 0.05), while H2O2 concentration, aberrant chromatin condensation, and abnormal spermatozoa were higher in the high THI group (P < 0.05). Blastocyst rates were significantly higher when low THI samples were used (P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher (P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher (P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development.

Practical methods to assess the effects of heat stress on the quality of frozen-thawed Belgian Blue semen in field conditions.

The increased exportation of semen and embryos of double-muscled beef breeds to tropical and developing countries makes it important to investigate the reproductive capacity of these breeds in adapting to tropical conditions. The aim of this study was to evaluate the quality of Belgian Blue semen collected after there is heat-stress (HS; as a mimic of tropical condition) compared with non-heat stressed (NHS; as their comfort zone), using practical spermatozoa staining methods such that prevail in developing countries. There was screening of semen kinetics using CASA and evaluation of their DNA-, acrosome, plasma membrane-integrity, and mitochondrial activity. For each staining technique, there was evaluation of 12 frozen-thawed semen samples from six Belgian Blue bulls collected after there were HS and NHS conditions in Belgium. Mixed linear regression models were used to assess the effects of HS for each CASA variable and staining method outcome using the replicate nested with bull as a random effect. There were differences (P < 0.05) in values when there were semen collections following HS and NHS conditions for several post-thawing kinetic variables. Furthermore, the mean percentages of DNA-, acrosome-, and plasma membrane-integrity, as well as mitochondrial activity were greater (P < 0.05) when semen was collected following NHS compared with HS conditions. Conclusively, results indicated that when there was collection of semen following HS conditions, there were detrimental effects on the viability and quality of Belgian Blue semen which is an important consideration for the semen collection, processing, and evaluation in tropical countries.

Influence of Different Combinations of Permeable and Nonpermeable Cryoprotectants on the Freezing Capacity of Equine Sperm.

This study was aimed to evaluate the effect of permeable cryoprotectants in combination with trehalose or sucrose on the freezing capacity of stallion sperm. For this purpose, the ejaculates (n = 24) were collected from four healthy mature Turkmen stallions. The ejaculates were pooled and diluted with one of the extenders containing a combination of 5% of permeating (dimethylacetamide [DMA]; dimethylformamide [DMF] or glycerol) and 50 mM of nonpermeating cryoprotectant agents (CPAs) (sucrose or trehalose) to a final concentration of 200 × 106 spermatozoa/mL. The extended samples were cryopreserved and thawed using a standard protocol. The samples were evaluated for motion kinetics, morphological abnormalities, plasma membrane functionality (PMF), viability, and lipid peroxidation. The results showed that the sperm cryopreserved in extender containing DMA produced higher (P ≤ .05) total motility, straightness, straight line velocity, curvilinear velocity, and lower (P ≤ .05) lipid peroxidation (malondialdehyde [MDA] concentration) compared with DMF and glycerol groups. Overall, both DMA and DMF have shown higher (P ≤ .05) sperm motion kinetics, viability, PMF, and lower (P ≤ .05) morphological abnormalities and MDA concentration compared with the glycerol. However, except morphological abnormalities, all of the other parameters did not differ between trehalose and sucrose. Likewise, there was no interaction between permeating and nonpermeating CPAs (P ≥ .05) except in terms of sperm abnormalities (P ≤ .05). In conclusion, the use of DMA or DMF as alternative CPAs of glycerol could be more effective for successful cryopreservation of stallion sperm. The nonsignificant interaction between permeating and nonpermeating CPAs for most of the post-thaw sperm parameters negates possible synergism among these compounds.

Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen.

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 µM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 µM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm.

Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm.

The present study aimed to determine the effect of different concentrations of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on post-thaw stallion sperm quality. The ejaculates collected from four healthy mature Turkmen stallions were pooled and divided into eight aliquots. The samples were diluted with extenders containing different concentrations (0.5, 1 or 2mM/mL) of BHA or BHT. The positive control (PC) samples were diluted with extender containing 0.5% ethanol (v/v) whereas; the negative control (NC) samples were diluted with basic extender only. Semen samples were frozen according to a standard protocol. After thawing of samples, sperm motility, viability, membrane integrity, total abnormality and lipid peroxidation were assessed. The greatest (P<0.05) values for total sperm motility, viability and plasma membrane functionality and least values for malonedialdehyde (MDA) concentration were observed in samples supplemented either with 1mM BHT or 2mM BHA. However, the progressive motility was greater (P<0.05) only in samples treated with 2mM BHA. In conclusion, the use of 1mM BHT or 2mM BHA in extender improves the freezing capacity of stallion sperm by reducing oxidative stress during freeze-thaw process.