Follicular dynamics in synchronized Italian Mediterranean buffalo cows

The aim of this study was to evaluate the length and the characteristics of the oestrous cycle in Italian Mediterranean buffalo cows, undergone synchronization of ovulation. The trial was performed on 32 buffaloes synchronized by the Ovsynch Program, which consists of an injection of GnRH on day 0, PGF2α on day 7 and GnRH on day 9. Starting on day 10 (Day 0 of the new cycle). Buffaloes undergone ultrasound examination of the ovaries on alternate days until the following heat. Follicular growth and corpus luteum formation and dimensions were recorded as well as the number of follicular waves. Statistical analysis was performed by ANOVA. Four animals (12.5%) did not show signs of oestrous and were excluded from the trial. The mean length of the oestrous cycle was 23.7±3.4 days. In particular, 1 animal (3.6%) showed an oestrous cycle characterized by 1 follicular wave with a length of 16 days, 17 subjects (60.7%) showed 2 follicular waves with a cycle length of 22.4±2.3 days and 10 buffaloes (35.7%) showed 3 follicular waves with a cycle of 26.8±2.0 days. These results confirm previous reports performed in buffalo species, although the cycle resulted longer in the 3-waves group

Detection of Brucella abortus DNA and RNA in different stages of development of the sucking louse Haematopinus tuberculatus

Background: Brucellosis is considered the world’s most widespread zoonotic infection. It causes abortion and sterility in livestock leading to serious economic losses and has even more serious medical impact in humans, since it can be a trigger to more than 500,000 infections per year worldwide. The aim of this study was to evaluate the role of Haematopinus tuberculatus, a louse that can parasitize several ruminants, as a new host of brucellosis. Louse specimens were collected from seropositive and seronegative water buffaloes and divided in 3 developmental stages: adults, nymphs and nits. All samples were separately screened for Brucella spp. DNA and RNA detection by Real Time PCR. In particular, primers and probes potentially targeting the 16S rRNA and the Brucella Cell Surface 31 kDalton Protein (bcsp31) genes were used for Real Time PCR and buffalo β actin was used as a housekeeping gene to quantify host DNA in the sample. A known amount of B. abortus purified DNA was utilized for standard curve preparation and the target DNA amount was divided by the housekeeping gene amount to obtain a normalized target value. A further molecular characterization was performed for Brucella strain typing and genotyping by the Bruce-ladder, AMOS-PCR and MLVA assays. Data were statistically analysed by ANOVA. Results: Brucella abortus DNA and RNA were detected in all developmental stages of the louse, suggesting the presence of viable bacteria. Data obtained by MLVA characterization support this finding, since the strains present in animals and the relative parasites were not always identical, suggesting bacterial replication. Furthermore, the detection of Brucella DNA and RNA in nits samples demonstrate, for the first time, a trans-ovarial transmission of the bacterium into the louse. Conclusions: These findings identified H. tuberculatus as a new host of brucellosis. Further studies are needed to establish the role of this louse in the epidemiology of the disease, such as vector or reservoir

Synchronization of estrus and artificial insemination in buffalo heifers by using two different protocols.

In Italy, because of the application of the out of breeding season mating (OBMS) technique in buffalo species, there are some months during which few deliveries, and consequently scanty milk production, are present. Buffalo heifers are lesser sensible to the seasonality compared to pluriparous animals and hence can be inseminated also in a period characterised by increasing day length, increasing milk production in some periods of the year. Few studies have been carried out in order to verify the most efficient method for synchronization of estrus and artificial insemination (AI) in buffalo heifers. The aim of this study was to compare two methods of synchronization of estrus largely used in buffalo species. The trial was carried out on 146 Italian Mediterranean buffalo heifers 781±103 days old and characterised by a body weight of at least 380 kg. Animals were divided in two groups according to their age and body weight. Heifers in group A (n=67) were synchronized by using the Ovsynch-TAI Program, which consists of 12 g buserelin acetate on Day 0, 0.524 mg PGF2α on Day 7 and 12 g buserelin acetate on Day 9, whereas animals in Group B (n=68) received a double injection of 0.524 mg of PGF2α 12 days apart. Each heifer was inseminated twice, 16 and 40 h after the second injection of GnRH in Group A and 60 and 84 h after the second injection of PGF2α in Group B. On day 26 post-insemination heifers underwent trans-rectal ultrasonography examination, in order to assess pregnancies, that were confirmed on Day 45 by rectal palpation. Statistical analysis was performed by chi-square test. Synchronization rate was similar in the two groups (93.1 vs. 91.9, respectively in Group A and B). No differences in terms of pregnancy rate were recorded between the two treatments. A higher although not statistically significant pregnancy rate was observed in Group B, which showed also a lower incidence of early embryo mortality. Similar results in terms of pregnancy rate in buffalo heifers have been previously reported. In conclusion both protocols can be used in order to synchronize buffalo heifers and concentrate the deliveries in some periods of the year

Influence of insemination technique and ovulation time on fertility percentage in synchronized buffaloes

The efficiency of superficial vs. deep insemination, and the influence of time of ovulation in relation to implant removal, were studied in 337 buffalo cows (after 96 h) and 187 heifers (after 72 h). The animals were synchronized using either a PRID vaginal implant or Crestar, subcutaneously deposited into the furthest part of the tail. Animals that had not ovulated by the second day were inseminated over 2 consecutive days (72- and 96-h heifers, respectively). Buffaloes that ovulated after 96 h showed a lower fertility rate, and the presence of oestral mucus favoured the conception rate in heifers only. Conception rate was higher with deep insemination. No difference was observed in cows or heifers that ovulated upon insemination. The results confirm the high variability that distinguishes ovulation time in the buffalo species. The better fertility percentage obtained using deep insemination in animals that ovulated within 24 h from implant and the lack of difference between the two insemination techniques in animals that ovulated at the time of insemination, prove that sperm survival in the female genital tract is less than 24 h

Intravenous PGF2α administration at the time of artificial insemination in buffalo species: preliminary results.

The efficiency of artificial insemination (AI) in buffalo species is low, above all if the animals are inseminated when day length is increasing. It was recently demonstrated in cattle that the administration of a PGF2α analogous at the time of AI increased plasma progesterone concentration on day 6 post-insemination and pregnancy rate (PR) in repeat breeder bovines. The aim of this study was to evaluate the effect of a single intravenous administration of PGF2α on the day of AI in synchronized buffaloes. The trial was carried out on 116 multiparous Italian Mediterranean Buffaloes at 160±128 days in milk and 57 heifers 767±139 days old. The animals were synchronized by using the Ovsynch-TAI Program, which consists of 12 mcg buserelin acetate on Day 0, 0.524 mg PGF2α on Day 7 and 12 mcg buserelin acetate on Day 9. Each buffalo was inseminated twice, 16 and 40 h after the second injection of GnRH. On day of the first AI buffaloes were divided in two groups: animals in Group A received an intravenous administration of cloprostenol, a PGF2α analogue, while buffaloes in Group B did not receive any injection. On day 26 post-insemination buffaloes underwent trans-rectal ultrasonography examination, in order to assess pregnancies, that were confirmed on Day 45 by rectal palpation. Statistical analysis was performed by chi-square test. A higher, although not statistically significant, PR was recorded in Group A vs. Group B, both in pluriparous buffaloes and in heifers, on day 26 and 40 (Table 1). Further studies are needed in order to increase the number of animals and verify if the administration of cloprostenol will be able to significantly increase PR in buffalo species and reduce the phenomenon of early embryo mortality

La sincronizzazione dell’estro nella bufala: metodiche e risultati di una sperimentazione italiana.

L’efficienza dell’inseminazione strumentale (IS) nella specie bufalina è bassa, soprattutto se gli animali sono inseminati quando aumentano le ore di luce giornaliera. È stato recentemente dimostrato nella specie bovina che la somministrazione di un analogo delle PGF2α al momento della IS è responsabile di un aumento dei livelli plasmatici di progesterone al giorno 6 post-inseminazione e di un maggiore tasso di gravidanza in bovine repeat breeder. Scopo di questo studio è stato di valutare l’effetto di una singola somministrazione endovena di PGF2α al momento della IS in bufale sincronizzate. La prova è stata condotta su 356 bufale pluripare di razza Mediterranea Italiana con una distanza dal parto di 181,7±88 giorni e 57 manze con un’età di 767±139 giorni. Gli animali sono stati sincronizzati mediante l’Ovsynch-TAI Program, che consiste in 12 microgrammi di buserelin acetato (Receptal®, Intervet) al giorno 0, 0,524 mg di cloprostenolo (Estrumate®, Schering-Plough Animal health S.p.A.), un analogo delle PGF2α al giorno 7 e 12 microgrammi di buserelin acetato al giorno 9. Ogni bufala è stata inseminata 16 ore dopo la seconda iniezione di GnRH e 40 ore nel caso in cui non avesse ovulato. Nel giorno della prima IS (giorno 10), e cioè al momento dell’inseminazione, le bufale sono state equamente divise in due gruppi: gli animali del gruppo A (n=180) hanno ricevuto una somministrazione di cloprostenolo (Estrumate®, Schering-Plough Animal health S.p.A.), mentre le bufale del gruppo B (n=176) hanno ricevuto uno stesso volume di soluzione fisiologica (placebo). La diagnosi di gravidanza è stata effettuata al giorno 45 mediante esplorazione rettale. L’analisi statistica è stata eseguita utilizzando il test del chi-quadro. Una percentuale di gravidanza più elevata a 45 giorni è stata osservata nel gruppo A vs. il gruppo B negli animali adulti (46,7 vs. 30,7%; P<0,01). Anche nelle manze si è riscontrato un tasso di gestazione più alto (63,3 vs. 51,9), ma, forse a causa del basso numero di soggetti in prova, non è stata evidenziata significatività statistica. È possibile pertanto confermare che la somministrazione di cloprostenolo al momento della IS incrementi la percentuale di gravidanza nella specie bufalina. La differenza tra bufale e manze potrebbe dipendere dal fatto che è verosimile che una parte delle bufale era repeat breeder (condizione che a causa della promiscuità sessuale nella mandria bufalina non è facile accertare). Se tale ipotesi è plausibile, si riconferma quanto riscontrato nelle bovine repeat breeder per le quali si è osservato un miglioramento della percentuale di gravidanza quando si somministra la PGF2α al momento della IS e si intravede nella short luteal phase o in un inadeguato sviluppo del corpo luteo la causa della minore efficienza della IS nella specie bufalina a conferma di precedenti riscontri. Altre spiegazioni del fenomeno devono essere ricercate nell’azione luteotrofica esercitata sulle cellule della teca cui si accennava in precedenza, oppure in una migliore sincronizzazione dell’ovulazione che si verifica negli animali trattati. In tal senso è stato, infatti, dimostrato il ruolo fondamentale che rivestono le prostaglandine, la cui concentrazione all’interno del follicolo preovulatorio è responsabile della deiscenza. È ipotizzabile che, soprattutto nella specie bufalina, caratterizzata da un’ampia variabilità del periodo fine calore-ovulazione, una migliore sincronizzazione della stessa favorisca considerevolmente la fertilizzazione dell’oocita

Superovulatory response to FSH+LH treatment with or without progesterone supplementation in buffalo.

The multiple ovulation and embryo transfer (MOET) technique represents an attempt to further contribute to genetic improvement. Several authors reported a scanty recovery rate in terms of embryos in buffalo species after superovulatory treatment, with an average of 1.43, 2.38 and 1.4-3.1 embryos/flushing/donor. The aim of this study was to verify if the presence of progesterone during the first two days of a FSH-LH superovulatory treatment may improve embryo recovery. The trial was carried out on 14 multiparous Italian Mediterranean Buffaloes at 113±98 days in milk from December to May. The animals were synchronized by a double injection of 0.524 mg of PGF2α 12 days apart. After 60 hours from the last injection of PGF2α buffaloes undergone clinical examination of the genital tract in order to evaluate estrus status: animals with tonic uterus and a follicle higher than 1.2 mm, with or without mucus vaginal discharge, were considered in estrus and a progesterone releasing intravaginal device was inserted (Day 0). Buffaloes were, hence, divided in two groups, according to parity and days in milk. In Group A (n=7) PRID was removed on Day 8, whereas in Group B (n=7) it was left till day 10. Both groups received a 4-day decreasing dosage of an equal mixture of 500 I.U. of FSH and LH since day 8. On day 10 all treated buffaloes were injected twice 12 hours apart with 0.524 mg PGF2α. Two artificial inseminations were performed 60 and 84 h after the second injection of PGF2α. Six days after the first insemination buffaloes undergone a uterine flushing with phosphate buffered saline supplemented with 1% fetal calf serum: each uterine horn was washed with at least 500 ml of solution. Embryos were immediately searched by filtration and vitrified, according to Neglia et al. (2001). Statistical analyses was carried out by t Student test. Two animals (one for each treatment) did not result responsive to the superovulation treatment. A higher, although not statistically significant, number of embryos was recorded in Group B vs. Group A if all animals are considered. However, if the analysis is carried out only on responsive buffaloes, a higher (P<0.05) recovery rate was recorded in Group B vs. Group A. These results are in accordance with previous reports in buffalo species. Progesterone supplementation during the first two days of the superovulation treatment seems to enhance the number of recovered embryos, although further studied are needed in order to apply the MOET in buffalo

Follicular dynamics in synchronized Italian Mediterranean buffalo cows.

The aim of this study was to evaluate the length and the characteristics of the oestrous cycle in Italian Mediterranean buffalo cows, undergone synchronization of ovulation. The trial was performed on 32 buffaloes synchronized by the Ovsynch Program, which consists of an injection of GnRH on day 0, PGF2alfa on day 7 and GnRH on day 9. Starting on day 10 (Day 0 of the new cycle). Buffaloes undergone ultrasound examination of the ovaries on alternate days until the following heat. Follicular growth and corpus luteum formation and dimensions were recorded as well as the number of follicular waves. Statistical analysis was performed by ANOVA. Four animals (12.5%) did not show signs of oestrous and were excluded from the trial. The mean length of the oestrous cycle was 23.7±3.4 days. In particular, 1 animal (3.6%) showed an oestrous cycle characterized by 1 follicular wave with a length of 16 days, 17 subjects (60.7%) showed 2 follicular waves with a cycle length of 22.4±2.3 days and 10 buffaloes (35.7%) showed 3 follicular waves with a cycle of 26.8±2.0 days. These results confirm previous reports performed in buffalo species, although the cycle resulted longer in the 3-waves group