ASSESSMENT OF MITOCHONDRIAL DNA COPY NUMBER AND FUNCTIONAL CHARACTERISTICS FOR ASSOCIATION WITH SEMEN QUALITY IN BUFFALO BULLS ACROSS DIFFERENT SEASONS
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Date
2022
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ICAR-NDRI, KARNAL
Abstract
Semen quality and quantity are affected by seasonal changes, particularly heat stress having
significant influence. Mitochondrial DNA (mtDNA) copy number has been utilized as a
measure of sperm quality in several species including mice, dogs, humans, cattle and buffalo.
The present study was carried out to correlate the mitochondrial DNA copy number, cellular,
biochemical parameters and gene expression data. Based on the semen quality data (HOST,
acrosomal integrity, viability) available across the seasons (winter, hot humid, hot summer and
comfort seasons), buffalo bulls were classified into seasonally affected (SA) and seasonally
non-affected (SNA) by heat stress. The semen samples collected from 8 target animals during
winter (Dec 21 – Jan 22) and hot summer (May 22 – June 22) seasons, were used for the study.
Fresh semen quality parameters (volume, mass motility and sperm concentration) and postthaw
motility of frozen semen of the SA and SNA bulls across summer and winter seasons,
were not significantly different (p<0.05). However, fresh semen collected in the winter season
had better seminal attributes in both SA and SNA bulls compared to the summer season.
Mitochondrial DNA copy number estimation was done by real-time PCR using a mitochondrial
coded gene (CYTC) and nuclear-coded gene (ACTB). The mean (±SD) number of
mitochondrial DNA copy number for all the animals was 13±2 and had a range from 8 to 18,
without any significant variation between the groups. Mitochondrial membrane potential
(MMP) values were significantly different for SA animals across the winter and summer
seasons. The variation in superoxide levels however was not significant in the animals across
the seasons. The dead acrosome reactive (DR) and the live acrosome intact (LI) values showed
significant variation across the summer and winter seasons in the SA group of animals. Semen
collected in the summer from SA bulls had the least live sperms with intact acrosomes. GSH
levels in the seminal plasma were not significantly different across the seasons and animal
groups. Catalase concentration in the seminal plasma however was significantly different
across the winter and summer seasons in SA animals. There was also no significant variation in
lipid peroxidation levels as determined by the malondialdehyde (MDA) marker, irrespective of
the seasons and the group of animals. The oxidative stress genes -ATF4, ATF5 and CYTC
have shown a 5-fold, 11-fold and 6-fold increase in the expression in the seasonally nonaffected
groups in the winter season. Fold changes of apoptosis-related genes BAK1 and
MCL2, were 4-fold and 6- fold respectively higher in Sthe NA group in winter compared to
summer.
In conclusion, the mitochondrial membrane potential (MMP), intact live acrosome (LI), and
Catalase levels were positively correlated with semen quality of selected parameters in the
fresh as well as frozen semen of Murrah buffalo bulls. There favourably high expression of
oxidative stress and anti-apoptotic genes in seathe sonally non-affected group in hot summer
compared to affected bull