Novel approaches in andrology examination and follicular fluid biochemical characterization in the optimization of reproductive technologies in farm animals

The purpose of the studies reported in this Thesis was to increase our understanding of two aspects of the reproductive system in farm animals: the andrological evaluation and the follicular fluid composition. The final aim was to give some tools that can be helpful in optimizing the application of assisted reproductive technologies. Studies were conducted in bulls, rams, alpacas, lamas and dairy buffalo cows. The first study was designed to investigate the efficacy of scrotal thermographic monitoring in the evaluation of young bulls with low semen production after GnRH administration. Yearling bulls with low semen quality were selected. Scrotal surface temperature (SST) and serum testosterone (T) were evaluated before and after administration of 10.5 µg Buserelin Acetate IV. Bulls were divided in two groups: LowTemp bulls had a decreased SST at 60 minutes; HighTemp bulls had an increased SST. After 60 minutes from Buserelin administration, LowTemp bulls had higher T values compared to HighTemp bulls: 14.32 ± 0.53 ng/ml vs. 10.30 ± 1.37 ng/ml respectively (mean ± SEM) (P<0.05). Reproductive performances in both groups improved after Buserelin administration, but the improvement was more marked in LowTemp bulls. In conclusion a decreased SST after GnRH administration was associated with better testicles functionality. The second study was done to assess the predicting value of trans-scrotal ultrasonography (TSUS) and testicular fine-needle aspiration cytology (TFNAC) on sperm production in rams. TSUS and TFNAC were performed on eighteen adult rams at the beginning of the breeding season. After evaluation, semen was collected twice with electroejaculation from each ram, and then epididymal spermatozoa were collected after slaughtering. Ultrasonography appearance of testicular parenchyma was analyzed to create a four classes division of the animals. Relative percentages of spermatogenic cells and Spermatic Index (SI = percentage of spermatozoa on the total of spermatogenic cells) were calculated after obsersation of TFNAC slides. Rams having a better echogenicity had a higher SI and a higher total sperm and number of straws per ejaculate either with electroejaculation or epididymal spermatozoa than the worst groups. Several cytological parameters were correlated with sperm production: the most interesting correlation was between SI and number of straws obtained by epididymal collection (0.667; P=0.003. In conclusion, TSUS and TFNAC can be usefully integrated in breeding soundness evaluation of rams, as these techniques had a potential as a marker of semen production. The third study documents the variability of follicular fluid (FF) composition of South American camelids, in different stages of ovarian activity. In the first experiment, ovaries of alpacas were collected at slaughterhouse and FF was aspirated dividing sample on the base of ovarian cycle stage and follicular dimension. Follicle size affected FF composition, either by biochemical analysis or electrophoresis. Conversely, the influence of corpus luteum was minimal. In the second study, ovaries of thirty lamas were scanned ultrasonographically to identify wave emergence. FF was aspirated trans-vaginally at three different stage of development (growing, static and regressing phase). The electrophoresis analysis revealed differences between groups in 4 classes of weight (250 KDa, 75 KDa, 25 KDa, 15 KDa). This study offers new information concerning the biochemical composition of follicular fluid in South American camelids, suggesting that oocytes grow and mature in an environment that changes in function of the follicle stage, while is minimally influenced by the presence of the corpus luteum. The aim of the forth study was to characterize the biochemical and fatty acids composition of the follicular fluid (FF) in water buffalo. Ovaries of buffalo were collected after slaughtering during breeding season. Fluid was aspirated from follicles of small and large follicles ( 6 mm respectively) and dividing females in those in luteal or follicular phase of estrous cycle. Biochemical analysis and gas chromatography were performed. Biochemical and fatty acids compositions were influenced by both follicular dimension and phase of ovarian cycle. Biochemical composition and its variation was in agreement with previously study conducted in buffalo and other species. Twenty-two fatty acids were identified in FF; nine were saturated fatty acids, six monounsatured fatty acids and seven polyunsatured fatty acids. The most dominant fatty acids were linoleic acid, oleic acid, palmitic acid, stearic acid and arachidonic acid. All the identified fatty acids concentrations varies at least because of follicle dimension or phase, with the exception of γ-linoleic acid and arachidonic acid which concentrations remain stable in all classes. The fifth study was designed to investigate the effect of different diets on the testicular fine-needle aspiration cytology (TFNAC) in rams. Eighteen rams were fed with three different diets since 8 months of age up to 1 year. The diets were: 1- pasture (N=6); 2- hay and commercial concentrate (N=6); 3- hay, commercial concentrate and a supplementation of conjugated linoleic acid (CLA) containing equal parts of cis-9, trans-11 and trans-10, cis-12 isoforms (N=6). After slaughtering, testes were collected to perform TFNAC. No statistical differences were observed in live weight and testicular weight between groups. CLA supplemented rams had higher relative percentage of early spermatogenic cells (primary spermatocytes and AB spermatids) and lower percentage of CD spermatids and SI compared to other groups. Total CLA concentration in diets was similar in pasture and CLA supplemented groups, but rams graze at pasture receive almost all cis-9, trans-11 isomer. Rams fed with hay and concentrate received a lower quantity of CLA in the diet. In conclusion, CLA supplementation and in particular the trans-10, cis-12 isomer affected negatively the TFNAC results in peripubertal rams probably because it returded the onset of puberty.Lo scopo degli studi riportati in questa Tesi è di aumentare le nostre conoscenze su due aspetti della biotecnologia applicata alla riproduzione negli animali da reddito: la valutazione andrologica e la composizione del fluido follicolare. Il fine è stato quello di fornire degli strumenti che possono essere utili nell’ottimizzazione della gestione riproduttiva. Gli studi sono stati condotti su tori, arieti, alpaca, lama e bufale. Il primo studio è stato creato per investigare l’efficacia del monitoraggio termografico scrotale nella valutazione di giovani torelli con scarsa produzione di seme, dopo somministrazione di GnRH. Sono stati selezionati torelli di circa un anno di età con scarsa qualità seminale. Sono stati valutati, prima e dopo la somministrazione di 10.5 µg di Buserelina, la temperatura superficiale dello scroto (SST) e il testosterone sierico (T). I tori sono stati divisi in due gruppi in funzione della risposta termica alla somministrazione di Buserelina: tori LowTemp con una diminuzione di SST a 60 minuti dal trattamento; tori HighTemp con un aumento della SST. Dopo 60 minuti dalla somministrazione di Buserelina, i tori LowTemp esibivano livelli di T maggiori rispetto a quelli dei tori HighTemp: 14.32 ± 0.53 ng/ml vs. 10.30 ± 1.37 ng/ml rispettivamente (media ± SEM) (P