Equine Spermatogenesis: Meiotic Chromosome Behavior and Recombination

Studying the spermatogenesis of horse is beneficial for the horse industry by identifying the causes of chromosomal abnormalities, which cause embryonic loss, congenital abnormalities and infertility. Little is known about the spermatogenesis in horse. This is the first report that investigates the horse spermatogenesis in detail, particularly metaphase I (MI) and prophase I (PI) stages of the first meiotic division. Meiotic recombination is considered to be the major outcome of meiosis. It is essential for proper chromosome segregation and formation of normal haploid gametes. Analysis of recombination frequency and distribution are crucial for genomic and association studies. Any alteration of the recombination frequency and positioning can cause non-disjunction and generation of aneuploidy. The frequency and distribution of chiasmata were estimated at MI chromosomes from fourteen fertile stallions. The average frequency of autosomal chiasmata was 49.45 ± 2.07, corresponding to a genetic length of 2,472.5 cM. All autosomal bivalents had at least one chiasma. The majority of chromosomes have one or two chiasmata, which are mostly distally localized. The frequency and the distribution as well as the genetic length of chiasmata were also estimated for the first time in eight different individual autosomes. Immunofluorescent localization was used to characterize the early stages of the first meiotic division as well as to examine the frequency and the distribution of DNA mismatch repair protein MutL Homologous Protein 1 (MLH1) foci on synaptonemal complexes (SCs) from sex fertile stallions. The mean frequency of autosomal recombination foci was 50.11±2.35. All autosomal bivalents had at least one recombination focus. In general, foci were located near the distal ends with some foci interstitially distributed. The distribution of MLH1 foci indicated positive interference; however, foci were very close to one another in rare instances. The average SCs relative length was highly correlated with the average number of MLH1 foci. MLH1 have been proposed to mark crossover sites at PI since the frequency and distribuation of MLH1 foci closely correspond to the frequency and distribution of chiasmata on MI chromosomes. iii | P a g e Spermatozoa viability, which include spermatozoa head and tail membrane integrity, acrosomal integrity and mitochondrial function assessment are the main sperm analysis parameters considered in this thesis to evaluate the stallion fertility using epididymal collected semen samples. The mean percentage of spermatozoa with viable heads and tails, using Chicago sky blue stain, was 81.26 ± 5.06. FITC-Pisum sativum agglutinin (FITC-PSA) and MitoTracer green were used successfully to assess the spermatozoal acrosomal status as well as the mitochondrial function, respectively. The mean percentage of spermatozoa with integrated acrosome was 93.85 ± 1.9, while for functional mitochondria was 95.63 ± 1.63. In conclusion, this finding is the cornerstone to understanding the genetic basis of normal horse spermatogenesis. Simultaneous assessment of different functional sperm parameters as well as investigating the synapsis and recombination frequency and distribution, at PI or MI, would assist with predictions of stallion fertility prior to breeding. In addition, this study will enable investigators to use linkage analysis in identifying and localising different genetic loci associated with specific traits