Abstract
Optimization of semen cryopreservation protocol for the production of high-quality semen dose
and development of accessible and low-cost sorting techniques for sex-linked spermatozoa for
obtaining optimized fertility is still an active area Of research, Therefore, the main objectives Of this
research were to produce high quality cryopreserved semen doses and to establish dextran swim-
up mothod for sox-linked Spermatozoa _ Semen was collected from six crossbred bulls (two
Brahman crcuss, two Sahiwal cross and two Jersey cross) and evaluated by observing in vitro
qualities and in Vivo fertility. For Vitro qualities viability, plasma membrane (PM) integrity,
sperm normality, total and progressive motility (POMT) and kinematics using a computer assisted
semen analyzer (CASA) were assessed for all experiments. One handmade extender TRIS-Citric
acid-Fructmse extender was used as control (Group T). Vitamin E enriched egg yolk (Group EE)
and Tocopherol with omega III enriched egg yolk (Group TP) were used for cryopreservation.
Quantification of sex-related spermatozoa in separated semen fractions using RFLP PCR along
with in vivo fertility and calf sex were studied. In vivo fertility was assessed by inseminating
crossbred cows at natural estrus. Experiment I results revealed highly significant (p<O.01)
variation in volume, concentration and mass activity and %viable sperm (p<O.05) parameters
among crossbred bull semen. The study showed the highest concentration, mass activity and
sperm with functional PM integrity, total motility and progressive motility in the semen of
Brahman cross, semen volume and viable sperm in Sahiwal crossbred, and normal sperm in Jersey
crossbred bulls. A high significant bræd differences was also found in straight line
velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL) and beat cross frequency
(BCF). Biochemical analysis of seminal plasma demonstrated substantial breed differences in total
cholesterol (TC) magnesium (Mg), zinc (Zn) and high density lipoprotein (HDL). Pearson's
correlation test revealed significant (p<O.05) positive relation among total motility, progressive
motility, seminal plasma (SP) glucose, Mg and TC, and negative association among straightness
(SIR), PM and Ca; between VCL and TG. Experiment II revealed the highest percentage of live
spermatozoa, hypoosmotic swelling test (HOST) positive spermatozoa, total motility and
progressive motility in the semen of Group TP. Whereas, the post-thaw sperm kinetics VSL VAP,
VCL, BCF and ALH (altitude of lateral head deviation) were highest in Group EE. Statistical
analysis revealed significant (p<O.01) dilution effect on in vitro sperm qualities Group T
and Group TP or between Group T and Group EE. This experiment also found significant (p<0.01)
breed effect only in Group T. The highest in vivo fertility was observed with frozen-thawed
Sahiwal crossbreed semen in all diluent groups. By collecting four fractions after swim up
separation, Experiment Ill showed that the viable spermatozoa and HOST positive spermatozoa of
different fractions were found to be significantly (p<0.05) lower than fresh semen. The swim-up
prcxedure improved normal spermatozoa percentage, total motility and progressive motility
percentage in different fractions, except Fraction 4. It was found that swim up techniques
inconsistently and non significantly (p>0.05) altered the kinetics in different fractions. In
Experiment IV, densitometric quantification of RFLP-PCR bands using Mylmage Analysis!
software revealed a more or less equal amount of sex specific chromosomes and calf sex in fresh
whole semen. We recorded the highest amount of Y chromosomes bearing spermatozoa in
Fraction 1 and of X chromosome bearing spermatozoa in Fraction 4 with significant differences
among fractions (p<O.01). A similar pattern in the amount of X and Y bearing were
observed in all three crossbred bulls. Jn vivo calving revealed significantly (p<0.01) higher
percentage of male calves in Fraction 1 and of female calves in Fraction 4 in this investigation.
Results suggest that Tris-based bull semen extender incorporated with omega III fatty acid-
enriched egg yolk and 1% tocopherol can be successfully used to obtain better post-thaw sperm
quality and in vivo fertility. In addition, in vivo male and female calves produced by fraction sorted
sperm of different bulls indicate modified swim-up technique as a useful alternative for sex-
selective sperm production in cattle.
Key words: Seminal trait, kinetic velocity, cryopreservation, swim-up, in vivo fertility, in vivo cal