Semen collection and semen preservation were carried out at PEARL-FUNAAB Poultry Breeding Center Laboratory of Animal Physiology Department, respectively at the Federal University of Agriculture, Abeokuta, Nigeria. The University is located on latitude 7010´ N, longitude 302´ E and at altitude of 76m above sea level. It lies within the South – West region of Nigeria having a tropical climate, with a mean annual rainfall of about 1,037mm and average temperature of 34.70C (Google Earth, 2019).
Experimental animals and management
Thirty cocks of 52 weeks old and three different breeds were used for this study. The cocks comprise of 10 FUNAAB Alpha (Normal feather), 10 Arbor Acre and 10 Dominant black. The animals were reared intensively in battery cages individually. They were fed with breeder mash concentrate feed with ad libitum supply of drinkable water
Semen collection and cryopreservation
Semen samples were collected into a graduated eppendorf tube using the abdominal massage method. Ejaculate with more than 80% motility were pooled for FUNAAB Alpha and exotic chickens and were diluted using two-fractioned tris-egg yolk extender as earlier described by Daramola et al. (2016). To do this, the semen was diluted in the Fraction 1 solution after which the Fraction 2 solution containing 7% glycerol (v/v) was subsequently added to fraction 1 at a ratio of 1:1. The diluted semen was then loaded into 2ml eppendorf tubes, sealed with polyvinyl and cooled gradually in a refrigerated incubator at a rate of 0.25oC/min and thereafter equilibrated at 4oC for 10 min. The straws were then placed in a canister at 4cm above liquid nitrogen for 10mins for equilibration before plunging them into the liquid nitrogen for 24 hours.
Thawing and semen evaluation
After 24 hours of cryopreservation, the frozen semen samples were retrieved and thawed in Clifton water bath (Model: 74178 by Nickel Electro Ltd, Weston-S-Mare Somerset, England) at 370C for 30mins. All microscopic evaluations were done using Celestron PentaView microscope (LCD-44348 by RoHS, China) at 400x magnification.
Sperm motility was determined as using Bearden and Fuquay (1997) procedure. Thawed semen samples of 5µl were placed directly on a pre-warmed microscope slide and overlaid with a 22 x 22mm cover slip. Each semen sample was measured using different slides (Two slides per sample). Ten microscopic fields were examined for each slide to observe progressively motile spermatozoa and the mean of the ten evaluations were expressed as a percentage of the final motility.
Sperm plasma membrane integrity
Sperm membrane integrity was determined using hypo- osmotic swelling test (HOST) assay as described by Zubair et al. (2013). For this assay, 10µl semen was incubated in hypo- osmotic solution (9g fructose and 4.9g sodium citrate/100ml distil water) at 370C for 30 minutes. 0.1ml of the mixture was spread over a pre-warm slide, covered with a cover slip and observed under an LCD digital microscope (400X). Two hundred spermatozoa were counted and intact sperm membrane were recorded as the percentage of spermatozoa with curly tail
Sperm livability and abnormality
Eosin-negrosin smear was used in evaluating sperm livability and abnormalities as described by Bearden and Fuquay (1997). A thin smear of mixture of semen and eosin-negrosine solution was drawn across the slide and dried. The percentage of morphologically abnormal spermatozoa with defects in the head, mid piece and tail were observed under LCD microscope (400x). Spermatozoa that appeared white and unstained under the microscope were recorded as live spermatozoa while those with eosin-negrosin stain are counted as dead spermatozoa.
Sperm acrosome integrity
The percentage of spermatozoa having intact acrosome was determined according of Ahmad et al., (2014) procedure. 50µl of each semen sample was added to a 500 µl formalin citrate solution (96ml 2.9 % sodium citrate, with 4 ml 37% formaldehyde). A small drop of the mixture was placed on a microscope slide and a total of 200 spermatozoa were counted in at least three (5) different microscopic fields for each sample. Intactness of acrosome was recorded as spermatozoa with normal apical ridge.
After 24 hours, Malondialdehyde (MDA) concentration was measured in thiobarbituric acid reactive substances (TBARS) according to Pipan et al. (2014). For this assay, 0.1 mL of sperm suspension was incubated with 0.1 mL of 150 mM Tris-HCl (pH 7.1) for 20 min at 37 °C. Subsequently, 1 mL of 10 % trichloroacetic acid (TCA) and 2 mL of 0.375 % thiobarbituric acid was added and incubated in boiling water for 30 min. Thereafter, it was centrifuged for 15 min at 3000 rpm inside blank tube and the absorbance was read with UV spectrophotometer (SW7504 model by Surgifriend Medicals, England) at 532 nm. The concentration of MDA was calculated as follows:
MDA (nmol/mL) = AT – AB/1.56 × 105
Where AT = the absorbance of the sample,
AB = the absorbance of the blank, 1.56 × 105 molar absorptivity of MDA.
Data obtained was subjected to Multivariate analysis using SAS 2002 and means was separated using Duncan Multiple Range Test. STEPDISC procedure was used to determine the variables that contributed to differentiation among the three breeds of chicken while CANDISC procedure was used to obtain canonical variables, canonical coefficients and Mahalanobis distance among the three breeds based on the selected traits. The DISCRIM procedure was used to obtain the percentage of correct assignment of each bird to its breed.