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Mayorga Munoz, Isabel Margarita (2009) Biotechnologies of reproduction: some alternatives to apply superovulation and embryo transfer in sarda sheep. [Tesi di dottorato]

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Abstract (inglese)

First experiment :

“FSH-p superovulatory treatment without progestagen sponges”

The aim of this study was to evaluate the superovulation response to FSH-p treatment without the use of intravaginal progestagen sponges in attemps to reduce the use of hormones and cost of procedures in a MOET programme. Twenty animals were divided into 2 groups: natural oestrus (NT) (n=10) and single sponge (SS) 40 mg FGA for 12 days (n=10) (control group).
Superovulatory treatment per sheep consisted of 350 I.U. of porcine FSH administered in eight decreasing doses at every 12 h starting on day 4 after onset of oestrus (day 0) in the NT group and 48 h before sponge removal in the SS group. A single dose of 125 µg (i.m) cloprostenol was injected on day 6 after oestrus detection in the NT group to induce ovulation. Contemporary to FSH treatment follicular development was assessed by ultrasonography. Ewes were naturally mated 24 h after cloprostenol injection or sponge removal in the NT and SS groups respectively.
Seven days after mating, inguinal laparotomy was performed and the number of corpora lutea (CL) was recorded. Embryos were recovered surgically by flushing each uterine. The recovered embryos were evaluated and scored on a scale of 1 to 3. Embryos with a score of 1 were considered of high quality. Data on number of corpora lutea (CL), embryos recovered (ER), embryos fertilized (EF) and high quality embryos (EQ1) per ewe were analysed by ANOVA. Recovery (RR), fertility (FR) and high embryo quality (Q1R) rates per treatment by a Chi Square analysis.
The ovulation response classes (medium vs high) and embryo quality rates (Q1, Q2 and Q3) per treatment were analyzed by Fisher’s Exact Test. In order to assess the effect of a presence of a large (presumptive dominant) follicle a Linear Model, Chi Square, Pearson Correlation and Linear regression procedures were applied to the variables in study.
Among all variables analysed results show statistical differences only in mean number of CL/ ewe (10.7±3.4 vs 7±3.2) and FR (100% vs 80%) (p<0.05) between NT and SS groups respectively. In conclusion it is possible to superovulate ewes avoiding the use of intravaginal sponges during the FSH-p treatment and some variables (CL/ewe; FR%) seem to be improved as well as costs and length of treatments are reduced.

Second experiment :

“Using the open-pulled straw as catheter (OPS-C) to symplify the embryo transfer technique with vitrified embryos”

The aim of this study was to test the efficiency of the OPS-catheter embryo transfer technique as an alternative to increase the use embryo transfer program in different farm conditions looking at cost- effective and less time consuming procedures.
Twenty one recipients were selected by oestrus detection with vasectomized rams and divided into 2 groups: OPS-catheter (OPS-C) (n=11) and TomCat (TomCat) (control group).
Due the fact that, the number of experimental animals was reduced, only embryos of NT group (Exp. 1) were considered for transfer. Among all recovered embryos (N°72) after flushing of NT group 52 were vitrified but only 22 were utilised for transfer in the OPS-C group, while, 20 fresh embryos were utilised for transfer in the TomCat group. Embryos were transferred in pairs by inguinal laparotomy into D7 synchronised recipient (D7: day 7 after natural onset of estrus) directly after thawing or after morphological evaluation in the OPS-C and TomCat groups respectively.
After a Chi Square analysis pregnancy rates (82% vs 70%), embryo survival rates ( 59% vs 60% ), twinning rates (44.4% vs 71.4 % ) and birth weight at lambing (2.8 ±0.8 vs 2.7±0.4) did not show any statistical differences between OPS-C and TomCat groups respectively.
The results in this experiment demonstred that, embryo transfer with the OPS as catheter is possible avoiding the use of microscope and expertise technicians. Thus, with this technique we decrease the cost of procedures and increase the use of embryo transfer in different farm conditions.


Abstract (italiano)

Primo esperimento :

“Trattamento di superovulazione FSH-p senza l’utilizzo di spugne contenenti progesterone”

Lo scopo di questo studio e stato quello di valutare la risposta superovulatoria al trattamento con FSH-p senza l’utilizzo di spugne intravaginali contenenti progesterone per cercare di ridurre l’uso di ormoni e il costo delle procedure in un programma MOET. Venti animali sono stati pressi in considerazione e divisi in due gruppi: trattamento con estro naturale (NT) (n=10) e singola spugna (SS) 40 mg FGA per 12 giorni (n=10) (gruppo controllo).
Il trattamento di superovulazione per ogni pecora consisteva di 350 Unità Internazionali di FSH suino somministrate in otto dosi decrescenti ogni 12 ore a partire dal giorno 4 dopo l’inizio dell’estro (giorno 0) nel gruppo NT e 48 ore prima della rimozione della spugna nel gruppo SS. Una singola dose di 125 µg (i.m) di cloprostenolo e stata iniettata nel giorno 6 dopo il rilevamento dell’estro nel gruppo NT per indurre l’ovulazione. Contemporaneamente al trattamento di FSH lo sviluppo follicare e stato valutato mediante ultrasonografia.
Le pecore sono state accopiate mediante monta naturale 24 ore dopo l’iniezione di cloprostenolo o la rimozione della spugna sia nel gruppo NT che nel gruppo SS rispettivamente.
Sette giorni dopo l’accoppiamento, e stata eseguita una laparatomia inguinale dopo di che il numero dei corpi lutei e stato segnato. Gli embrioni sono stati recuperati chirurgicamente mediante flushing in ogni corno utero. Gli embrioni recuperati sono stati valutati e classificati con un punteggio in scala da 1 a 3.
Gli embrioni con voto 1 sono stati considerati di alta qualità. I dati riguardanti il numero dei corpi lutei (CL), degli embrioni recuperati (ER), degli embrioni fertilizzati (EF), e gli embrioni di alta qualità (EQ1) per ogni pecora sono stati analizzati con ANOVA. I tassi di recupero (RR), fertilità (FR) e qualità embrionale (Q1R) per trattamento con un’analisi Chi Square. La classe di risposta ovulatoria (media vs alta) e tassi di qualità embrionale (Q1, Q2 and Q3) per trattamento sono stati analizzati con il test Fisher’s Exact.
Per valutare l’effetto della presenza di un largo (presunto dominante) follicolo sono stati applicate per le variabili studiate un Modello Lineare, Chi Square, Correlazione Pearson e procedure di regressione Lineare.
Tra tutte le variabili analizzate i risultati hanno dimostrato differenze statistiche solo nella media del numero dei CL/pecora (10.7±3.4 vs 7±3.2) e FR (100% vs 80%) (p<0.05) tra i gruppi NT e SS rispettivamente. In conclusione è possibile superovulare pecore evitando l’utilizzo di spugne intravaginali durante il trattamento di FSH-p e alcune variabili (CL/pecora; FR%) sembrano essere migliorate e anche ridotti i costi e la lunghezza dei trattamenti.

Secondo esperimento:

“Utilizzo dell’ open-pulled-straw come catheter (OPS-C) per semplificare la tecnica di trasferimento degli embrioni vitrificati”

Lo scopo di questo studio è stato di testare l’efficienza della tecnica di trasferimento embrionale con l’utilizzo dell’ OPS-catheter, come alternativa, per incrementare l’uso del trasferimento embrionale nelle più svariate condizioni aziendale, con un occhio di riguardo a procedure più semplici, veloci ed effettivamente economiche.
Ventuno riceventi sono state selezionate attraverso il rilevamento dell’estro con arieti vasectomizzati e divise in due gruppi: OPS-catheter (OPS-C) (n=11) e Tom Cat (TomCat) (gruppo controllo).
Considerato che, il numero degli animali nell’esperimento era ridotto, solo gli embrioni del gruppo NT (Esp.1) sono stati reclutati per il trasferimento. Tra tutti gli embrioni recuperati (N°72) dopo il flushing del gruppo NT 52, sono stati vitrificati, ma solo 22 sono stati utilizzati per il trasferimento nel gruppo OPS-C, mentre, 20 embrioni freschi sono stati utilizzati per il trasferimento nel gruppo TomCat. Gli embrioni sono stati trasferiti in coppia attraverso una laparatomia inguinale in riceventi sincronizzate al giorno7 (D7: giorno 7 dopo l’inizio dell’estro naturale) direttamente dopo lo scongelamento o dopo valutazione morfologica nei gruppi OPS-C e TomCat rispettivamente.
In seguito all’analisi Chi Square dei tassi di gravidanza (82% vs 70%), tassi di sopravvivenza embrionale (59% vs 60% ), tassi di gemellarità (44.4% vs 71.4 % ) peso degli agnelli nati (2.8 ±0.8 vs 2.7±0.4), non è stata dimostrata nessuna differenza statistica tra i gruppi OPS-C and TomCat rispettivamente.
I risultati in questo esperimento hanno dimostrato che, il trasferimento embrionale con OPS-catheter è possibile evitando l’uso del microsopio e l’impiego di tecnici esperti. In questo modo diminuendo il costo della procedura è possibile incrementare l’utilizzo dell’ embryo transfer anche in aziende in diversi condizioni e scarse possibilità economiche.

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Tipo di EPrint:Tesi di dottorato
Relatore:Morgante, Massimo
Correlatore:Stelletta, Calogero - Dattena, Maria
Dottorato (corsi e scuole):Ciclo 19 > Corsi per il 19simo ciclo > MEDICINA DEGLI ANIMALI
Data di deposito della tesi:29 Gennaio 2009
Anno di Pubblicazione:2009
Parole chiave (italiano / inglese):alternatives procedures, superovulation, embryo transfer, sheep
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/08 Clinica medica veterinaria
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Cliniche Veterinarie
Codice ID:1628
Depositato il:29 Gen 2009
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