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Vencato, Juri (2014) Novel approaches in andrology examination and follicular fluid biochemical characterization in the optimization of reproductive technologies in farm animals. [Tesi di dottorato]

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Abstract (inglese)

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 and > 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.

Abstract (italiano)

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<0.05). Le performance riproduttive di entrambi i gruppi sono migliorate in seguito alla somministrazione di Buserelina, tuttavia il miglioramento è stato più marcato nei tori LowTemp. In conclusione, una diminuzione della SST dopo la somministrazione di GnRH è associata a una migliore funzionalità testicolare.

Il secondo studio è stato eseguito per accertare il valore predittivo dell’ecografia trans-scrotale (TSUS) e della citologia testicolare per aspirazione con ago sottile (TFNAC) sulla produzione spermatica negli arieti. TSUS e TFNAC sono state eseguite su diciotto arieti adulti all’inizio della stagione riproduttiva. Dopo la valutazione, il seme è stato raccolto mediate elettroeiaculazione per due volte da ciascun ariete, e gli spermatozoi epididimali sono stati raccolti dopo la macellazione. L’aspetto ecografico del parenchima testicolare è stato analizzato per creare una classificazione in quattro classi degli animali. Le percentuali relative delle cellule spermatogenetiche e l’indice spermatico (SI = percentuale di spermatozoi sul totale delle cellule spermatogeniche) sono stati calcolati dopo la lettura dei vetrini di TFNAC. Arieti con una migliore ecogenicità testicolare sono risultati avere un più alto SI e un maggior valore di spermatozoi totali e numero di dosi inseminanti prodotte per eiaculato sia per elettroeiaculazione che spermatozoi epididimali rispetto ai gruppi peggiore. Alcuni parametri citologici sono stati correlati con la produzione spermatica: la più interessante correlazione trovata è quella tra lo SI e il numero di dosi ottenute tramite raccolta epididimale (0.667; P=0.003). In conclusione, TSUS e TFNAC possono essere utilmente integrati nella valutazione della breeding soundness degli arieti, poiché queste tecniche sono potenzialmente degli indici di produzione spermatica.

Il terzo studio documenta la variabilità della composizione del fluido follicolare (FF) nei camelidi Sud Americani, in diversi stadi di attività ovarica. Nel primo esperimento, ovaia di alpaca sono state raccolte al macello e il FF è stato aspirato dividendo i campioni sulla base dello stadio dell’attività ovarica e alla dimensione follicolare. Le dimensioni follicolari influenzano la composizione del FF, sia la composizione biochimica sia quella elettroforetica. Diversamente, l’influenza della presenza del corpo luteo è minima. Nel secondo studio, le ovaie di trenta lama sono state valutate per via ecografica per individuare l’emergenza follicolare. Il FF è stato aspirato per via transvaginale a tre diversi stadi di sviluppo (fase di crescita, statica e di regressione). L’analisi elettroforetica ha rivelato delle differenze tra i gruppi in quattro classi di peso molecolare (250 kDa, 75 kDa, 25 kDa, 15 kDa). Questo studio fornisce nuove informazioni sulla composizione biochimica del FF nei camelidi Sud Americani, suggerendo che gli oociti crescono e maturano in un ambiente che varia in funzione dello stadio follicolare, mentre è solo minimamente influenzato dalla presenza del corpo luteo.

Lo scopo del quarto studio era di caratterizzare la composizione biochimica degli acidi grassi del fluido follicolare (FF) di bufala. Ovaia di bufala sono state raccolte dopo la macellazione avvenuta all’interno della stagione riproduttiva. Il fluido è stato aspirato da follicoli di piccole e grandi dimensioni (< 6 mm e > 6 mm rispettivamente) e dividendo gli animali in quelli in fase follicolare e fase luteale del ciclo. Sono state eseguite un’analisi biochimica e una gas cromatografia. La composizione biochimica e degli acidi grassi sono influenzate sia dalle dimensioni follicolari che dalla fase del ciclio ovarico. La composizione biochimica e le sue variazioni sono in accordo con gli studi precedenti condotti su bufale e altre specie. Sono stati identificati ventidue acidi grassi nel FF; nove sono acidi grassi saturi, sei sono acidi grassi monoinsaturi e sette poliinsaturi. Gli acidi grassi più rappresentati sono l’acido linoleico, acido oleico, acido palmitico, acido stearico e acido arachidonico. Le concentrazioni di tutti gli acidi grassi identificati variano in funzione di almeno una dimensioni follicolari o fase del ciclo, ad eccezione dell’acido γ-linoleico e dell’acido arachidonico le cui concentrazioni rimangono stabili.

Il quinto studio è stato creato per investigare l’effetto di diverse diete sulla citologia testicolare per aspirazione con ago sottile (TFNAC) negli arieti. Diciotto arieti sono stati alimentati con tre diete differenti dagli 8 mesi all’anno di età. Le diete sono state: 1- pascolo (N=6); 2- fieno e un mangime commerciale (N=6); 3- fieno, mangime commerciale e integrazione di acido linoleico coniugato (CLA) contenente parti uguali d’isomero cis-9, trans-11 e isomero trans-10, cis-12 (N=6). Dopo la macellazione, i testicoli sono stati raccolti per eseguire una TFNAC. Non sono state osservate differenze statistiche nel peso vivo e nel pero dei testicoli tra i gruppi. Gli arieti che hanno ricevuto l’integrazione di CLA, avevano percentuali relative di cellule precoci della spermatogenesi (spermatociti primari e spermatidi AB) più elevate e percentuali inferiori di spermatidi CD e indice spermatico rispetto agli altri gruppi. La concentrazione totale di CLA nelle diete era similare nei gruppi al pascolo e integrato con CLA, ma gli arieti al pascolo hanno ricevuto quasi unicamente l’isomero cis-9, trans-11. Gli arieti alimentati con fieno e mangime hanno ricevuto una minore quantità nella dieta. In conclusione, l’integrazione di CLA e in particolare dell’isomero trans-10, cis-12 influenza negativamente i risultati della TFNAC in arieti peripuberi probabilmente perché ritardano l’inizio della pubertà.

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Tipo di EPrint:Tesi di dottorato
Relatore:Stelletta, Calogero
Dottorato (corsi e scuole):Ciclo 26 > Scuole 26 > SCIENZE VETERINARIE
Data di deposito della tesi:27 Gennaio 2014
Anno di Pubblicazione:31 Gennaio 2014
Parole chiave (italiano / inglese):andrology/andrologia; thermography/termografia; testicular cytology/citologia testicolare; follicular fluid/fluido follicolare; bulls/tori; rams/arieti; South American camelids/camelidi sudamericani; buffalo cows/bufale
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/10 Clinica ostetrica e ginecologia veterinaria
Struttura di riferimento:Dipartimenti > Dipartimento di Biomedicina Comparata ed Alimentazione
Codice ID:6403
Depositato il:03 Nov 2014 13:06
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