Laparoscopy of the genitourinary tract of small ruminants

Laparoscopic endosurgery is an important tool for small ruminant breeding especially due to its wide versatility, both in reproductive biotechnologies and therapeutic approach. The purpose of this review is to present the applicability and advantages of endosurgery for routine use in small ruminants. Several endosurgical techniques of the genitourinary tract of small ruminants, including artificial insemination, embryo transfer, and oocyte retrieval for IVF, ovariectomy, cystotomy and renal biopsy are approached. The endoscopic techniques can improve the development of potential zootechnical indexes on the short-term period in comparison to traditional approaches.

Efficiency of superstimulatory protocol P-36 associated with the administration of eCG and LH in Nelore cows

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos.

Expression of estrus modifies the gene expression profile in reproductive tissues on Day 19 of gestation in beef cows

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Reproductive parameters of dairy goats submitted to estrus synchronization with prostaglandin F2a associated or not to hCG at estrous onset

Coodernação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES)

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Validation of a new proposal of quantitative sperm culture for industrialized bull semen

The Brazilian Ministry of Agriculture (MAPA) has discussed the mandatory culture of industrialized semen, both to ensure biosefety, and to prevent in vitro fertilization problems caused by oocyte contamination with ubiquitous and opportunistic bacteria from preputial microbiota. Pour plate, a quantitative technique recommended by the World Organization for Animal Health (OIE), is operationally difficult and costly for routine analysis in Artificial Insemination Centers (AICs). The objectives of this study were to evaluate and validate viable superficial bacteria counts (VSBC), in CFU/mL, compared with pour plate counts, in industrialized bull semen samples from AICs. Semen straws from Projeto Hungria - MAPA bulls were used. VSBC and pour plate were carried out in parallel in serial dilutions of the samples, from 10(-1) to 10(-5). CFU/mL means or medians recorded in each dilution and technique were compared, and no statistical differences were observed between the two techniques regarding the quantification of bacteria in CFU/mL, suggesting that it may be possible to replace pour plate for CBSV, a cheaper and more practical technique.

Evaluation of Inductively Coupled Plasma Mass Spectrometry for Determining Ca, Cu, Fe, Mg, Mn, Se and Zn in Bovine Semen Samples using a Simple Sample Dilution Method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 mu L) were diluted 1:50 in a solution containing 0.01% v/v Triton (R) X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 mu g L-1 for Ca-44, Cu-63, Fe-57, Mg-24, Zn-64, Se-82 and Mn-55, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Evaluation of inductively coupled plasma mass spectrometry for determining Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen samples using a simple sample dilution method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 µL) were diluted 1:50 in a solution containing 0.01% v/v Triton® X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 µg L-1 for 44Ca, 63Cu, 57Fe, 24Mg, 64Zn, 82Se and 55Mn, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone, equine chorionic gonadotropin, or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n ¼ 36) and anestrous (n ¼ 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P ¼ 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n=8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n=5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5×108 tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion=12.5 %), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion= 100 %). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc- Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.

Ruolo del Porcine Circovirus tipo 2 (PCV2) nella patologia riproduttiva del suino

La tesi è organizzata in 4 capitoli: -nel primo vengono brevemente riferite le patologie associate all’infezione da PCV2 con particolare riferimento all’iter diagnostico ed al ruolo rivestito dall’esame istologico e dalla identificazione dell’agente eziologico in situ contestualmente alle lesioni istologiche; -nel secondo viene presentato un iter diagnostico originale da applicare in condizioni di campo, qualora si voglia accertare la presenza del PCV2 nei tessuti dei prodotti di natimortalità/aborto del suino. In specifico si riferisce all’applicazione del protocollo in 2 aziende ed i risultati vengono analizzati per una revisione critica del protocollo impiegato; -nel terzo vengono presentati i risultati di un protocollo di infezione con PCV2 per via genitale tramite seme infetto. Scrofe convenzionali sono state sincronizzate per l’estro e fecondate con un’unica dose di seme PCV2 negativo alla PCR (gruppo controlli) o sperimentalmente esposto al PCV2 (gruppo infette). I risultati vengono analizzati in funzione delle ripercussioni che l’infezione precoce in gravidanza può produrre sulla scrofa (mancata gravidanza, ritorno in calore), sui feti e sugli invogli fetali. Viene stabilito il ruolo protettivo degli anticorpi circolanti al momento dell’infezione, stante l’evenienza che un basso titolo anticorpale si associa a viremia prolungata e maggiore numero di feti positivi al virus; -nel quarto viene presentato un esperimento sovrapponibile a quello riferito nel capitolo 3, però con la presenza anche di un gruppo di soggetti convenzionali vaccinati ed infettati con PCV2 durante la fecondazione artificiale usando seme sperimentalmente esposto al virus. Nella discussione dei risultati vengono enfatizzati 2 aspetti importanti nell’epidemiologia dell’infezione da PCV2: la eliminazione di virus è fortemente ridotta dalla vaccinazione, con conseguenze verosimilmente positive sulla circolazione del virus negli effettivi dell’allevamento; l’esposizione uterina è protetta dalla vaccinazione, stante la bassa percentuale di placente infette nel gruppo dei soggetti vaccinati rispetto a quelli non vaccinati e nei controlli.

Comparison of the effect of a CIDR-Select synch versus a long-term CIDR based AI protocol on reproductive performance in multiparous dairy cows in Swiss dairy farms

Background Synchronization programs have become standard in the dairy industry in many countries. In Switzerland, these programs are not routinely used for groups of cows, but predominantly as a therapy for individual problem cows. The objective of this study was to compare the effect of a CIDR-Select Synch and a 12-d CIDR protocol on the pregnancy rate in healthy, multiparous dairy cows in Swiss dairy farms. Methods Cows (N = 508) were randomly assigned to CIDR-Select Synch (N = 262) or 12-d CIDR (N = 246) protocols. Cows in the CIDR-Select Synch group received a CIDR and 2.5 ml of buserelin i.m. on d 0. On d 7, the CIDR insert was removed and 5 ml of dinoprost was administered i.m.. Cows in the 12-d CIDR group received the CIDR on d 0 and it was removed on d 12 (the routine CIDR protocol in Swiss dairies). On d 0 a milk sample for progesterone analysis was taken. Cows were inseminated upon observed estrus. Pregnancy was determined at or more than 35 days after artificial insemination. As a first step, the two groups were compared as to indication for treatment, breed, stud book, stall, pasture, and farmer's business using chi square tests or Fisher's exact test. Furthermore, groups were compared as to age, DIM, number of AI's, number of cows per farm, and yearly milk yield per cow using nonparametric ANOVA. A multiple logistic model was used to relate the success of the protocols to all of the available factors; in particular treatment (CIDR-Select Synch/12-d CIDR), milk progesterone value, age, DIM, previous treatment of the uterus, previous gynecological treatment, and number of preceding inseminations. Results The pregnancy rate was higher in cows following the CIDR-Select Synch compared to the 12-d CIDR protocol (50.4% vs. 22.4%; P < 0.0001). Conclusion The CIDR-Select Synch protocol may be highly recommended for multiparous dairy cows. The reduced time span of the progesterone insert decreased the number of days open, improved the pregnancy rate compared to the 12-d CIDR protocol and the cows did not to have to be handled more often.

Repression of common bull sperm flora and in vitro impairment of sperm motility with Pseudomonas aeruginosa introduced by contaminated lubricant

Semen collected from clinically healthy bulls at an artificial insemination centre was examined for bacterial diversity. While bacteria that are normally present in the common flora of bovine semen were absent, such as Mycoplasma sp., Proteus sp. and Corynebacterium sp., all semen samples contained an unusually high number of Pseudomonas aeruginosa strains. Analysis via pulsed field gel electrophoresis demonstrated that one particular P. aeruginosa strain, present in a sealed bottle of lubricant, was widespread in bull semen. This strain was shown to secrete substances that inhibited both the growth of bacteria constituting the normal bull sperm flora and the motility of spermatozoa in vitro. This study demonstrated that commercially available lubricants might contain bacteria that can spread amongst breeding bulls and affect the quality of semen. Bacteriological controls and species' identification are necessary at several production levels, including lubricants and extenders, to ensure high semen quality and avoid the spread of pathogens.

[Search for Neospora caninum DNA in bull semen using PCR ]

Neospora caninum represents one of the most frequent abortifaciant organisms worldwide. The parasite is diaplacentally transmitted from the pregnant cow to the fetus, where it normally leads to the delivery of a healthy, however persistently infected calf. Abortion thus is a relative rare event. The transmission of bovine neosporosis occurs in more than 90% of the cases vertically due to the endogenous reactivation of a persistently infected mother. Exogenous infections are therefore responsible for less than 10% of the cases.The question arises about which infection sources may be relevant in this context. In Switzerland, the role of dogs as definitive hosts has been shown to be of low significance in that respect. Recently, discussion focused on the potential of infectious bull semen following natural or artificial insemination. Thus, a few years ago a report documented the detectability of N. caninum-DNA in the semen of naturally infected bulls by nested-PCR. As a consequence, we decided to gain own experience by investigating 5 separate semen specimens per animal, originating from 20 N. caninum-seropositive bulls used for artificial insemination in Switzerland. All probes turned out to be negative by nested PCR. Based upon our laboratory experiences, the potential bull semen-associated Neospora-problem seems not to affect the Swiss bull population, thus there is no evidence to include further respective means of control.

Effect of Continuous Infusion of Relaxin on Progesterone, Oxytocin, and Relaxin Blood Concentrations and Time of Parturition in Beef Heifers

These studies were designed to determine whether continuous intravenous infusion of increasing dosages of porcine relaxin during late pregnancy in beef heifers would influence circulating blood concentrations of relaxin, progesterone, and oxytocin, and time of onset of parturition. Beef heifers were bred by artificial insemination and, on Day 277, fitted with indwelling jugular cannulas for hormone infusion and blood sampling from Day 277 to 286. Intravenous infusion of purified porcine relaxin (pRLX, 3000 U mg-1) was started in heifers (n = 8) at increasing dosages (200 U h-1 on Days 277 and 278, 300 U h-1 on Days 279 and 280, 500 U h-1 on Day 281, 600 U h-1 on Day 282, and 700 U h-1 on Days 283 to 286). Phosphate buffer saline (PBS, 10 ml h-1) was infused during these same times to control (n = 6) animals. Relaxin treatment steadily increased the circulating plasma concentration of immunoreactive relaxin to more than 120 ng ml-1 compared with less than 0.5 ng ml-1 in PBStreated controls. Relaxin infusion in increasing dosages over the treatment time was associated with a significant decrease (P < 0.01) in plasma progesterone concentration compared with the PBS controls. Plasma levels of oxytocin at 4- hour intervals remained similar (P > 0.05) during the pretreatment period and throughout continuous infusion of pRLX and PBS. Although continuous intravenous infusion of relaxin resulted in a decrease in circulating blood levels of progesterone, it did not significantly reduce the interval between the beginning of pRLX treatment and parturition compared with the PBS-infused control heifers. These results indicate that continuous intravenous infusion of high levels of porcine relaxin resulted in a decrease in progesterone secretion in late pregnant beef heifers.