The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Use of coenzyme Q-10 to improve the pregnancy rate in sheep.

One of the factors responsible for less pregnancy rates is the use of frozen semen in sheep due to the oxidative stress created by the process. The aim of this experiment was to test the effects of adding coenzyme Q-10 (CoQ10) to the seminal extender on sperm quality and the pregnancy rate of sheep. In this study, ejaculates from eight Dorper rams of reproductive age were used and tested in four treatments: Control (pure BotuBov®), C1 (175 µM of CoQ10), C3 (350 µM of CoQ10), and C7 (700 µM of CoQ10). Samples were collected in triplicate from each animal, and sperm analysis was performed by CASA after thawing at 0 h and 2 h. The samples were also analyzed by flow cytometry for plasma and acrosomal membrane integrity, stability, lipid peroxidation, mitochondrial potential, and superoxide anion production. In total, 198 ewes were inseminated by laparoscopy and divided into two groups: control (n=98) and C7 (n=100). Pregnancy diagnosis was performed at 30 days. Coenzyme Q10 proved to be safe for semen cryopreservation, not altering sperm kinetic values between the groups post-thawing. In flow cytometry, the C1 and C7 groups achieved a better index of plasma membrane integrity and membrane stability (P<0.05). A increased pregnancy rate was observed in C7 (52 %) compared to the control (38 %). In conclusion, coenzyme Q10 assists in the cryopreservation process, protecting the sperm cell and improving pregnancy rates in ewes.

Acute post-orchiectomy pain does not reduce alpha rams' interest in feed resources.

Sheep pain is an animal welfare issue monitored based on behavioral responses, including appetite. Dominant (alpha) males have priority for accessing limited feed resources, however, the effects of pain on feed interest in members of a group with defined social hierarchy are unknown. Our objective was to investigate effects of acute post-orchiectomy pain on alpha rams' interest in accessing a limited feed resource. Eighteen rams were randomly housed in pens of 3 rams. After acclimation, the first 5-d (consecutive) battery of a behavior test was performed. In this test, 180 g of the regular diet concentrate was placed in a portable trough in the center of the pen; this feed was supplemental to the diet and represented a limited, albeit strongly preferable feed resource. Rams were filmed for 5 min after the feed introduction. Hierarchical levels (alpha, beta, and gamma) were defined based on the social hierarchical index according to higher initiator and lower receptor agonistic behaviors from the social network analyses. After 15 d, a second 5-d behavioral test battery was repeated. On the following day, alpha rams were castrated. Flunixin meglumine was given immediately before surgery and a final behavioral test was performed 8 h post-orchiectomy, concurrent with an expected peak in postoperative pain. For all recordings, the latency, frequency, and duration of time that each ram had its mouth inside the feed trough were recorded, and the Unesp-Botucatu sheep acute pain scale pain scale (USAPS) was applied. The social hierarchical index was highest in alpha rams, followed by beta and gamma. The pain scores were statistically equivalent across the 11 evaluation days for beta and gamma rams, whereas there was an increase in the final evaluation for alpha. There was no difference in latency, frequency, and duration between alpha, beta, and gamma rams across evaluations. We concluded that acute post-orchiectomy pain did not decrease alpha rams' interest in accessing limited feed. Routine feeding offers a valuable chance to detect pain-related behavior using the USAPS in rams. However, dominance may confound appetite-related behaviors in assessing acute pain, as alpha rams' interest in limited feed remained unaffected by the pain.

The effect of progesterone length in timed AI in ewes.

This study aimed to evaluate the reproductive performance of ewes submitted to timed AI protocol with 7 (D7) or 9 (D9) days of progesterone. A total of 220 crossbred ewes (Doper × Santa Ines) were distributed in the treatments according to body weight (52.67 ± 11.76 kg), body condition score (2.5 ± 0.8; scale of 1-5), and the female category (multiparous, nulliparous and primiparous). Ewes received an intravaginal device of 0.3 g of progesterone (CIDR® Easy breed) on day 0, keeping in ewes for 7 or 9 days, which were the experimental treatments. At the time of P4 withdrawal, ewes received 300 IU of eCG (Novormon®) and 6.70 mg of dinoprost tromethamine (Lutalyse®). Insemination by laparoscopy was performed between 46 and 59 h after device removal, lasting from 1 to 5 min/ewe, which did not differ between protocols. The estrus detection rate was higher in the 9-day protocol compared to the 7-day protocol (82% and 65%, respectively, P = 0.0096). However, the 7-day protocol tended to increase pregnancy rate on the TAI compared to the 9-day protocol (45% and 33%, respectively, P = 0.09). The plasma concentration of progesterone at device removal was higher in the 7-day protocol than in the 9-day protocol (2.35 and 1.22 ng/mL, respectively, P = 0.04). We conclude that reducing the length of the estrus synchronization protocol to 7 days is recommended, which improved the reproductive response in ewes.

Correction: Validation of the Unesp-Botucatu composite scale to assess acute postoperative abdominal pain in sheep (USAPS).

[This corrects the article DOI: 10.1371/journal.pone.0239622.].

Validation of the Unesp-Botucatu composite scale to assess acute postoperative abdominal pain in sheep (USAPS).

A scale with robust statistical validation is essential to diagnose pain and improve decision making for analgesia. This blind, randomised, prospective and opportunist study aimed to develop an ethogram to evaluate behaviour and validate a scale to assess acute ovine postoperative pain. Elective laparoscopy was performed in 48 healthy sheep, filmed at one preoperative and three postoperative moments, before and after rescue analgesia and 24 hours after. The videos were randomised and assessed twice by four evaluators, with a one-month interval between evaluations. Statistical analysis was performed using R software and differences were considered significant when p <0.05. Based on the multiple association, a unidimensional scale was adopted. The intra- and inter-observer reliability ranged from moderate to very good (intraclass correlation coefficient ≥ 0.53). The scale presented Spearman correlations > 0.80 with the numerical, simple descriptive, and visual analogue scales, and a correlation of 0.48 with the facial expression scale. According to the mixed linear model, the scale was responsive, due to the increase and decrease in pain scores of all items after surgery and analgesic intervention, respectively. All items on the scale demonstrated an acceptable Spearman item-total correlation (0.56-0.76), except for appetite (0.25). The internal consistency was excellent (Cronbach's α = 0.81) and all items presented specificity > 0.72 and sensitivity between 0.61-0.90, except for appetite. According to the Youden index, the cut-off point was ≥ 4 out of 12, with a diagnostic uncertainty zone of 4 to 5. The area under the curve > 0.95 demonstrated the excellent discriminatory capacity of the instrument. In conclusion, the Unesp-Botucatu pain scale in sheep submitted to laparoscopy is valid, reliable, specific, sensitive, with excellent internal consistency, accuracy, discriminatory capacity, and a defined cut-off point.

New Genotypes of Coxiella burnetii Circulating in Brazil and Argentina.

Coxiella burnetii, the zoonotic agent of Q fever, has a worldwide distribution. Despite the vast information about the circulating genotypes in Europe and North America, there is a lack of data regarding C. burnetii strains in South America. Here, we show the presence of novel multispacer sequence typing (MST) genotypes of C. burnetii in two clusters detected in Brazil and Argentina that seem to be distant in parenthood. Argentinian strains isolated from a tick belongs to a new phylogenetic branch of C. burnetii, and the Brazilians strains may be related to MST 20 and 61. Multilocus variable number tandem repeats analysis (MLVA) typing provided a deeper resolution that may be related to host clusters of bovines, caprine, ovine, and ticks. Our results corroborate with the reports of geotypes of C. burnetii. Thus, we highlight the need for more genotyping studies to understand the genetic diversity of C. burnetii in South America and to confirm the hypothesis of host-related genotypes. We also emphasize the importance of virulence studies for a better understanding of Q fever in the region, which may help in surveillance and disease prevention programs.