Use of Infrared Thermography during Ejaculation Process and Its Link with Semen Quality and Freezability in Dogs.

This study aimed to describe the thermal variation of external reproductive tracts during ejaculation in relation to sperm quality in dogs. Forty-six adult fertile dogs were monitored using a thermal camera before, during and after the semen collection, taking into account penile and scrotal temperatures as reproductive thermal patterns while eye and perianal temperatures were recorded as complementary thermal patterns of behavioral response. The parameters were classified depending on age (≤4 years and >4 years), body weight (BW) (≤75 kg and >75 kg), sperm concentration (CON) (≤300 million and >300 million), total testicular volume (TTV) (≤600 cm3 and >600 cm3) and total ejaculation time (TET) (≤800 s and >800 s) of the animals from which semen was collected successfully. Heavier males (p < 0.05) that have more consistent testicles (p < 0.01) as well as quicker ejaculate responders (p < 0.001) and lower scrotal temperature had better semen (Δ motility) freezability. The lower eye temperature prior to the ejaculation (p < 0.01), lower scrotal temperature following ejaculation (p < 0.01), and conversely, higher penile temperature during the ejaculation (p < 0.001) had a higher sperm concentration. Furthermore, the sperm freezability was negatively correlated with total ejaculation time (r = -0.39, p < 0.05) and sperm abnormalities were lower in the ejaculate of dogs having a higher temperature of the scrotum, bulbus and penis. In conclusion, infrared monitoring throughout semen collection in dogs can provide information on behavioral reactions during human manipulation, as well as semen quality and testicular functionality.

Companion animals get close to the toxic aspects of antropogenic world: cytotoxicity of phthalates and bisphenol A on dog testicular primary cells.

Phthalates, which are among the most abundant plasticizers, have detrimental effects on the reproductive system. Similar to human, dogs are prominently exposed to phthalates in daily routines at low concentrations; while toys, training devices and commercial dog foods are considered as the primary sources of exposure. This study aimed to reveal and compare the cytotoxic effects of selected phthalates (Benzyl butyl phthalate (BBP), Diethyl phthalate (DEP), Bis (2-ethylhexyl) phthalate (DEHP), Di-'isobutyl' phthalate (DIBP), Di-'isodecyl' phthalate (-DIDP) Di-'isononyl' phthalate (DINP), Dimethyl phthalate (DMP), Di-n-octyl phthalate (DNOP)), and Bisphenol A (BPA) following 24 h exposure on primary testicular parenchymal cells of dog in vitro at concentrations between 0.001 and 2.5 nM. According to cytotoxicity results, DEHP was found to be the most toxic phthalate with IC50 at 22.53 µM; while DMP was the least (169.17 nM). IC50 of BPA was 161.81 nM, less than the average (61.95 nM) of phthalates. In addition, dog primary testicular cells were found more susceptible to the high molecular weight phthalates (DNOP, DEHP, DINP, DIDP) than low molecular weight phthalates (DMP, DEP, DIBP, BBP). Further studies should focus on morphological, physiological and molecular differences to comprehend the mechanisms involved as well as decreasing the risk for impaired spermatogenesis caused by environmental toxicants in companion animal medicine.

Red pine (Pinus brutia Ten) bark tree extract preserves sperm quality by reducing oxidative stress and preventing chromatin damage.

This study aimed to investigate the effectiveness of using red pine bark tree extract (P; Pinus brutia Ten) as a TRIS extender in an attempt to prevent oxidative stress in bull spermatozoa after freezing. Semen specimens were obtained from Simmental bulls via an artificial vagina and pooled. They were separated into five specimens and diluted with Tris extender consisting of P (200, 100, 50 and 25 µg/ml) and P free (control; C) up to a final concentration of 16 × 106 per straw. All specimens were equilibrated for a period of 4 hr at a temperature of 4°C, following which they were filled in 0.25-ml French straws and frozen. Addition of P resulted in favourable tail length in comparison with C (p < .05). The lowest malondialdehyde levels and the highest glutathione levels were detected in all P groups (p < .05). Supplementation with P did not show advanced results in terms of total, progressive sperm motility and total abnormality in comparison with C (p > .05). In conclusion, it has been shown that although P added to a Tris extender does not have a positive effect on sperm motility, it prevents chromatin damage by reducing oxidative stress, in addition to reducing head abnormalities when used at the amount of 50 µg/ml.

Semen collection by trans-rectal digital stimulation and insemination campaign in goat.

The overall purpose of this study was to describe a method of semen collection via trans-rectal digital massage (TDM) and to carry out a related fertility trial in Angora goat. Sixteen Angora bucks (ranging 1-4 years) and 28 nulliparous does (1-2 years) were used in this study. Semen samples were collected via trans-rectal massage from 85.71% of the bucks in multiple attempts (18/21). The mean values of volume, pH, mass motility, total motility, concentration, viability, abnormal spermatozoa rate and ejaculation time were 0.64 ± 0.09 ml, 6.3 ± 0.21, 2.7 ± 0.34, 58.18 ± 5.1%, 3.68 ± 0.31 × 109 /ml, 71.38 ± 7.12%, 18.22 ± 2.48% and 3.4 ± 0.33 min respectively. Oestrus was detected with teaser buck and confirmed by using infrared thermography and ultrasonography (US). The success rate of synchronisation was found as 71.4% (20/28). On Day 21, pregnancy diagnosis was performed trans-rectally with US and the pregnancy rate was determined as 78.57% (11/14). TDM method of semen collection seems to be easily applicable to the buck and it could be a good alternative to collect semen as well as its use in artificial insemination campaign. Thermal monitoring is found to be a valuable tool to monitor the response to hormonal driven ovulatory synchronisation in Angora does during timed artificial insemination.

Gallic and carnosic acids improve quality of frozen-thawed ram spermatozoa.

The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen-thawed ram spermatozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris-based extender and divided into five equal portions containing: 0.05 or 2 mM of GA; 0.05 or 0.2 mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5 cm above liquid nitrogen for 12 min then plunged. Computer-aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2 mM GA had greater total motility than control spermatozoa (39.9 ± 3.01 vs. 29.2 ± 1.31%, mean ± SEM, p < .05). The PMAI was greatest in 0.2 mM CA (13.3 ± 0.68%), whereas HMMP was highest in 0.05 mM CA but lowest in control (22.9 ± 4.95 and 11.4 ± 3.64% respectively; p < .05). In conclusion, for cryopreservation of ram semen in Tris-based extender, supplementation with 2 mM GA increased post-thaw motility, whereas supplementation with 0.05 mM CA enhanced mitochondrial function.

Genomewide association of male reproductive traits in Aksaray Malakli dogs.

Male reproductive parameters are often used for the functional examination and evaluation of predicted genetic values for future aspects. However, these traits are relatively reliable until the measurable effects are expressed on desired traits. Therefore, we aimed to associate the single nucleotide polymorphism (SNP) genotype of the investigated characteristics and reproductive loci. A total of 46 male dogs are divided into three age groups (I ≤ 3 years, n = 19; II 4-6 years; n = 19, and III ≥7 years, n = 8). The testis, scrotum and body weight, libido sexualis and ejaculation time for each fraction were monitored as functional traits, while the pH, fractional semen volume, motility, concentration, and abnormal and dead spermatozoa rate were recorded as spermatological traits. The Affymetrix Canine 127 K SNP genotyping array v2 (Affymetrix Inc., California, USA) was used for SNP genotyping. In the primary results, the scrotal circumference was found to be higher in group II compared to other groups (p < 0.05) and the lowest total abnormal spermatozoa rate was found in group I (p < 0.05). The normal spermatozoa rate was found to be significantly above the threshold in relation to the SNP in chromosome 17. In conclusion, this study represents an exciting first step towards SNP association with dog semen spermatological parameters. Future studies might be undertaken to evaluate this SNP region for gene-knockout and expression analysis and for fine mapping to validate and/or discover the exact position of the effect region.

Effect of cholesterol loaded cyclodextrin on semen cryopreservation of Aksaray Malakli shepherd dogs of different ages.

The objective of the study was to determine the effect of cholesterol-loaded cyclodextrin (CLC) on the quality parameters of semen from Aksaray Malakli Shepherd dogs of different age groups. Forty-eight male dogs were divided into 3 groupings according to their ages (young age (Y): ≤3 years, n: 20; middle age (M): 4-6 years, n: 20; old age (O): ≥7 years; n: 8). The sperm-rich portion of the ejaculate from each dog was divided into four aliquots and extended with either tris as a control (C) or tris loaded with 0.5, 1.0, and 1.5 mg/120 × 106 CLC as low (L), intermediate (I), and high (H) doses, respectively. Following equilibration for at least half an hour, the straws were frozen in nitrogen vapor and then stored in liquid nitrogen at least for 48 h. Later, the frozen straws were thawed in a water bath for spermatological evaluation. Significant differences were observed between different age groups in terms of the spermatological parameters (p < 0.05). The evidence suggests that increasing age is associated with poor in-vitro spermatological parameters and CLC was able to protect the acrosome integrity from cryo-damage during the freeze-thawing process. Better semen freezability characteristics were obtained at young ages, considering the overall parameters.

The use of infrared thermography to detect the stages of estrus cycle and ovulation time in anatolian shepherd dogs.

BACKGROUND: The aim of the study is to evaluate the effectiveness of thermographic monitoring, using the temperature changes of perianal and perivulvar areas for the determination of estrus in Anatolian Shepherd bitches. Fifteen bitches were used in the study. Blood and vaginal smear samples were collected and thermographic monitoring of perianal and perivulvar areas were carried out starting from proestrus to early diestrus. Also, external signs of estrus were investigated. Smear samples were evaluated by light microscopy after Diff-Quik staining method and superficial and keratinized superficial cells were determined as percentage (S + KS%). Progesterone and luteinizing hormone measurements were done by radioimmunoassay. The difference in temperature between perianal and perivulvar areas was evaluated through thermographic images by FLIR ResearchIR Software. RESULTS: According to the results obtained from the study, differences between progesterone and S + KS% were statistically significant (P < 0,05). Although temperature showed increase and decrease with progesterone and S + KS%, the differences were not important statistically (P > 0,05). Serum luteinizing hormone levels did not sign any difference (P > 0,05). CONCLUSIONS: As a result, thermographic monitoring alone is not enough for estrus detection in Anatolian Shepherd bitches. However, it can be used to assist the actual estrus detection technique in terms of providing some foreknowledge by evaluating the differences in temperature.

Vulvar thermal pattern following synchronization of estrus is linked to fertility after timed artificial insemination in goat.

Aim of this study was to study the vulvar thermal pattern variation during the timed artificial insemination protocol in Angora goat and identify the relationship with the successful rate. Does (36 adult healthy females) were synchronized using PGF2α at the day 0, 11 days of progesterone impregnated sponges intra-vaginally, PMSG 48 h before sponges withdraw (day 11) and the intra-cervical inseminations were carried out 48 h later (Timed Artificial Insemination: TAI) with chilled semen. Vulvar (VST) and perivulvar (PST) areas were considered to evaluate the thermal pattern during the protocol at the day 0 and at the TAI using a thermo camera (E60, FLIR System). Differences of temperature (ΔT) between the surfaces were calculated for each time. The does were monitored for pregnancy, delivery time and prolificacy. Pregnant (P) and non-pregnant (NP) does were compared in terms of VST, PST and ΔT using two ways ANOVA considering time and pregnancy as sources of variability. VST was lower than PST in all the monitored does (P < 0.05) (34.79 ± 0.14 vs 36.58 ± 0.14 °C) and without differences between P and NP at day 0 (35 ± 0.18 vs 36.39 ± 0.22 °C). Significant difference (P < 0.05) between P and NP does was recorded at TAI in terms of VST (33.89 ± 0.31 vs 35.40 ± 0.24 °C) and ΔT (-3.16 ± 0.34 vs -1.62 ± 0.26 °C). In conclusion thermal emission by glabrous surfaces in goat may be used to identify the right response induced by hormonal treatments and to optimize the application of assisted reproductive techniques at the field level.