Semen Collection Method Research Articles

Effect of Semen Collection Methods on the Quality of Pre‐ and Post‐thawed Bali Cattle (Bos javanicus) Spermatozoa

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post-thawed semen quality. The collection methods employed were electro-ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM+EE). A total of 25 untrained Bali bulls (age between 2 and 4years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer-assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM+EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM+EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2-3 and >3-4years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM+EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)-based sexing and effective semen collection methods for black-headed and straw-necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black-headed and 4 straw-necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W-chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro-ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw-necked ibis using the massage method. Limited motility, viability and concentration of straw-necked ibis sperm were observed. The sperm length was 180μm and that of the nucleus was 30μm with acrosome located at the tip of the nucleus. Thus, the PCR-based sexing proved to be an accurate molecular sexing method for black-headed and straw-necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw-necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.

A novel method for semen collection and artificial insemination in large parrots (Psittaciformes)

The paper described a novel technique for semen collection in large psittacines (patent pending), a procedure which was not routinely possible before. For the first time, a large set of semen samples is now available for analysis as well as for artificial insemination. Semen samples of more than 100 psittacine taxa were collected and analysed; data demonstrate large differences in the spermatological parameters between families, indicating an ecological relationship with breeding behaviour (polygamous versus monogamous birds). Using semen samples for artificial insemination resulted in the production of offspring in various families, such as Macaws and Cockatoos, for the first time ever. The present technique represents a breakthrough in species conservation programs and will enable future research into the ecology and environmental factors influencing endangered species.

EVALUACIÓN BACTERIOLÓGICA DE SEMEN DE VERRACOS USADOS COMO REPRODUCTORES EN GRANJAS PORCINAS DE LA ZONA DE LURÍN, LIMA

The objective of the present study was to evaluate the bacterial content in semen from 30 healthy boars used as sires in pig farms in the coastal area of southern Lima (Lurin). Half of boars were from two technically advanced farms and the other half from one semi technically oriented farm. The gloved-hand method for semen collection was used and the collected semen was filtered to separate the gel fraction. The results indicated that in the technical farms, 73% of ejaculates (11/15) showed bacterial growth and 13% (2/ 15) exceeded the OIE limit of 5,013 UFC/ml, whereas in the non-technical farm , all ejaculates showed bacterial growth and 60% (9/15) exceeded the OIE limit. Pseudomona aeruginosa was the bacteria with higher number of colonies per semen sample and Escherichia coli was the most common bacteria.

Avaliação de dois métodos para condicionamento e coleta de sêmen em quatro espécies do gênero Mazama

O desenvolvimento de técnicas não invasivas para a obtenção de sêmen de cervídeos facilita a criação de bancos genômicos, que são importantes instrumentos para a conservação ex situ e in situ. Este trabalho teve como objetivo criar uma metodologia não-invasiva de coleta de sêmen e comparar duas técnicas de coleta em quatro espécies do gênero Mazama: M. americana, M. gouazoubira, M. nana e M. nemorivaga. Para tanto, foram utilizados seis machos (M) e duas fêmeas (F) da espécie M. americana, 3M e 2F de M. gouazoubira, 1M e 1F de M. nana e 2M e 1F de M. nemorivaga. Para cada técnica testada, foi realizado um período de habituação dos animais ao manejo. Em seguida, duas técnicas de condicionamento e coleta foram avaliadas. Na primeira delas foi utilizada uma fêmea em estro com desvio lateral do pênis para vagina artificial (FEDL), obtendo-se a coleta de 50% dos indivíduos (100% dos machos de M. gouazoubira e 50% dos machos de M. americana), não obtendo ejaculados das demais espécies. Na segunda técnica, utilizando um manequim taxidermizado com urina de fêmea em estro (MUFE) não foi possível a coleta de nenhum ejaculado. Em todas as fases foi observado o comportamento do macho quanto ao tempo de interesse e aproximação, reflexo de "Flehmen", ato de cheirar ou lamber, exposição do pênis, ereção, número de falsas montas, tentativas de cópula e ocorrência de agressividade entre os animais.

Improved semen collection method for wild felids: Urethral catheterization yields high sperm quality in African lions (Panthera leo)

For wild and domestic felids, electroejaculation (EE) is the most common semen collection method. However, the equipment is expensive, there is a risk of urine contamination and animals usually show strong muscular contraction despite general anesthesia. Accordingly, we tested the feasibility of a different approach using urethral catheterization (UC) in seven African lions, previously described for domestic cats only. After general anesthesia with the α2-agonist medetomidine (which also stimulates semen release into the urethra) and ketamine, a transrectal ultrasound was performed to locate the prostate. A commercial dog urinary catheter (2.6 or 3.3 mm in diameter) was advanced approximately 30 cm into the urethra to allow semen collection into the lumen of the catheter by capillary forces. After retraction, sperm volumes between of 422.86 ± 296.07 μl yielded motility of 88.83 ± 13.27% (mean ± SD) with a mean sperm concentration of 1.94 × 109/ml. Here we describe a simple, field friendly and effective method to attain highly concentrated semen samples with excellent motility in lions and potentially other wild felid species as an alternative to electroejaculation.

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl®, Andromed® and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1−/PI− (intact spermatozoa), and Mitotracker+/YO-PRO-1− (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl® and Andromed® resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl® or Andromed® are used as freezing extenders.

Semen collection and evaluation of captive coatis (Nasua nasua)

Semen samples (n=105) were collected through eletroejaculation from six adult male coatis (Nasua nasua) between January 2007 and December 2008 at Universidade Federal de Mato Grosso Zoo, Cuiabá, Brazil. Mean values were: volume (mL); concentration (sperm/mL); total motility (%); progressive sperm motility (scale, 0-5); live spermatozoa (%); acrossome integrity (%); primary defects (%); and secondary defects (%). There was high correlation between total motility and live sperm; total motility and progressive sperm motility; total motility and acrossome integrity; live sperm and progressive motility; live sperm and acrossome integrity and volume and concentration. The method for semen collection was considered safe and efficient. It can be used for the evaluation of breeding potential of coati in captivity and for the establishment of new assisted reproductive technology (ART) for threatened neotropical carnivores species.

Factors associated with failure in breeding soundness examination of Western USA rams

Breeding-soundness examination (BSE) and eradication of Brucella ovis infection in rams are critical components of flock-health programs. The aims of this retrospective, cross-sectional study were to describe the results of BSE in a large sample of rams in the Western USA and to determine the association between BSE outcome and the semen collection method (penis manually extended vs. retained in the preputial cavity), ram body-condition score (BCS), the presence of ulcerative posthitis, and the size of the flock of origin. We evaluated the first BSE in a given year for rams from Colorado, Wyoming, and Utah, USA, from 2000 through 2007. Breeding-soundness examination consisted of physical examination, scrotal circumference and BCS measurement, semen collection by electroejaculation, and microscopic examination of semen motility, morphology, and leukocyte concentration. We assigned a reason for failure to each failed BSE and used multivariable logistic and Poisson regressions to measure associations between ram and flock variables and the risk or reason for failure on BSE. A non-random, owner-selected subset of rams was tested for antibodies to B. ovis by serum indirect ELISA (iELISA). The Rogan–Gladen corrected B. ovis seroprevalence was measured.Of the 14,667 BSEs performed on 11,804 rams, 29.0% were classified as “failed;” the most common reason for failure was substandard semen parameters (43.8%). Breeding-soundness examinations were more likely to have been categorized as failure for inflammatory causes when performed on rams from medium-sized flocks (OR 1.6; 95% CI 1.1, 2.3) and large flocks (OR 1.4; 95% CI 1.0, 1.9) (P=0.02), suggesting that larger flocks are at higher risk of contagious diseases. The adjusted seroprevalence of B. ovis antibodies among tested rams in this study was 10.0%. Of 233 rams seropositive to B. ovis, 125 (53.6%) were subclinical, a finding that supports the importance of this test in ram BSE. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters (P<0.001), but ulcerative posthitis and the semen collection method were not (P=0.09 and 0.34, respectively). However, collection of semen with the penis retained in the preputial cavity resulted in greater odds of leukospermia relative to semen collection with the penis extended (OR 4.1; 95% CI 2.9, 5.9; P<0.001), presumably from contamination of the semen sample with preputial leukocytes. For ram BSE, therefore, semen collection with the penis manually extended from the sheath is recommended to limit leukocyte contamination of the sample.

Impact of Collection Method on Assessment of Semen HIV RNA Viral Load

BackgroundThe blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal) viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load.Methodology/Principal FindingsSemen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM) either undiluted (Visit 1) or directly into transport medium (Visit 2). Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples.Conclusions/SignificanceThe method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment.

Effect of semen collection method on sperm motility of gray wolves (Canis lupus) and domestic dogs (C. l. familiaris)

Genetic management of Mexican gray wolves includes semen banking, but due to the small number of animals in the population and handling restrictions, improvements in semen collection and cryopreservation rely on results from studies of domestic dogs. Semen collection from wolves requires anesthesia and electroejaculation, which introduce potentially important variables into species comparisons, as dog semen is typically collected manually from conscious animals. To investigate possible effects of collection method on semen quality, we compared semen collection by the traditional manual method and by electroejaculation (EE) in a group of dogs (n = 5) to collection by EE only in wolves (n = 7). Samples were divided into two aliquots: neat or diluted in Tris/egg yolk extender, with motility evaluated at intervals up to 24 h. There were no differences (P > 0.10) in sperm motility in either neat or extended samples at 24 h from EE dogs and wolves, although motility of the wolf neat samples declined more rapidly (P < 0.05). However, there were differences (P < 0.01) between EE and manually collected dog semen in motility at 24 h, in both the neat and extended samples. Therefore, general motility patterns of dog and wolf semen collected by EE were similar, especially when diluted with a Tris/egg yolk extender, but sperm collected from dogs by EE did not maintain motility as long as manually collected samples, perhaps related to the longer exposure of EE samples to more prostate fluid.

Non-invasive determination of fecal steroid hormones relating to conservation practice in giant panda (Ailuropoda melanoleuca)

Abstract The endangered giant panda is the flagship species of wildlife conservation, a status that results in heightened attention to their well-being. However, one factor that hinders the ability to monitor this species in the wild is the difficulty to measure relevant physiological parameters which quantify the survival status in conservation research. In this study, we employed a non-invasive sampling method and immunochemical assays to determine if measurement of fecal steroid hormones is a viable option in monitoring the sex ratios and stress levels of a giant panda population. The results indicate that 1) the yearly concentration of fecal testosterone is a possible biomarker for distinguishing gender, and 2) invasive methods of artificial insemination and semen collection as well as parturition, cause an increase in fecal cortisol concentration. This study suggests that the noninvasive sampling of feces can be a practical tool to monitor physiological stress in free-ranging and fenced giant panda populations, and could be useful in the forthcoming giant panda census in determining sex ratios in the wild.

Reproductive characteristics of captive greater rhea (Rhea americana) males reared in the state of São Paulo, Brazil

Rheas (Rhea americana) belongs to the ratite group. Considering the commercial significance of this birds, some techniques, such as semen collection, were standardized. In this study, 107 male rheas (3 to 4 years of age) reared in commercial farms in the state of Sao Paulo, Brazil, were used. Semen was collected during the breeding and off-breeding seasons of 2001, 2002, and 2003. Bird hierarchical behavior was observed. Birds were restrained performed using a box and a black hood. Semen was collected by digital pressure on the base of the phallus, which size was measured, and the presence or absence of spiral shape was observed. Immediately after collection, semen samples were evaluated for volume, motility, sperm concentration, and morphology. In a limited number of birds, blood samples were collected to measure testosterone levels. Among the 69 birds studied during the breeding season, 44 presented large phalluses, out of which 26 showed spiral shape. The method of semen collection was efficient. The following semen parameter results were obtained: volume (0.68 ±0.14 ml), motility (61.11±11.54%), sperm concentration (3.29±1.33 x109 sptz/ml), and number of spermatozoa per ejaculate (2.40±1.38x109 sptz/ml). Morphological abnormalities were analyzed and recorded. Testosterone levels were statistically different (p = 0.0161) between the breeding and non-breeding season (53.28±18.41 ng/ml and 5:57±3.81 ng/ml, respectively). Variations in phallus size were also found between the breeding and non-breeding seasons. Larger phalluses and higher testosterone levels were correlated with dominant behavior. The results of the present experiment confirmed that it is possible to collect semen from rheas, allowing the future use of biotechnologies such as artificial insemination.

Colheita e avaliação do sêmen de sagui-de-tufo-branco (Callithrix jacchus)

y of different extenders on the semen from common marmoset to improve the quality of physical and morphological characteristics. Three adult specimens were used and nine ejaculates were collected from each one, using electroejaculation. To break up the seminal clot, nine variations of three different extenders were tested. TALP, Ca+2 free, with 0.5% tripsin, during 45 minutes at 37oC, was the best extender, dissolving partially the semen clot. The electroejaculation is a viable semen collection method for common marmoset, obtaining success in all animals, but the semen quality was relatively poor. KEY WORDS: Callithrix jacchus, electroejaculation, primates, semen.

Comparison of AutoMate ® and the gloved-hand method for boar semen collection

Comparison of AutoMate ® and the gloved-hand method for boar semen collection

Seminal Plasma Composition from Ejaculates Collected by Artificial Vagina and Electroejaculation in Guirra Ram

This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10-25 kDa rank, the IMAGE ANALYSIS software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma.

Assessment of Fertility Status in the Male Sumatran Rhino at the Sumateran Rhino Sanctuary, Way Kambas National Park, Lampung

Sumatran rhino is the most endangered rhino species. Its population is estimated less than 300 individuals remaining in the wild with highly declining rate to 50% in the last 15 years. The number of male rhinoceroses in the captivity are very few, therefore the assessment of its fertility is very important in order to support the breeding success since the captive breeding success is very poor. The objectives of this study were (i) to determine the male reproductive status, (ii) to establish a reliable semen collection method, and (iii) to assess semen parameters of the fresh collected sample. Three methods of semen collection were examined to determine its fertilizing potential, i.e. (i) stimulated combination of artificial vagina (AV), penile massage (PM) and accessory gland massage (AGM); (ii) AV and PM; and (iii) only with PM. The first method gave the best result with an ejaculation success of 85.71% (6/7, n = 7). The second and third methods obtained an ejaculation success rate of 50% (2/4, n = 4)) and 25% (1/4, n = 4), respectively. The collected ejaculates had a volume of 1.2-12.4 ml with whitish to cream turbid colour and pH 6.90-6.99. Sperm concentration was (143-333) x 103 sperm/ml. The quality of the sperm was low with only approximately 1% of them moved forward slowly. Approximately 80% of the spermatozoa were immature (prox. cytoplasmic droplet) with head (macro-, microcephalic) and tail abnormalities (broken tail). Semen quality increased after several collections and the amount of immature sperm decreased up to 5%. Electroejaculation procedure could produce 34 ml semen, but no sperm was found in the ejaculate. Hence, the combination of AV, PM, and AGM could get higher volume of ejaculate compared to other methods, but sperm concentration was better obtained using AV and PM only. Repeated semen collection increased semen quality, although the male has low fertilizing capacity due to low sperm concentration (oligozoospermia) and small volume of the ejaculate (oligospermia). Key words: Sumatran rhino, semen, sperms, collection

Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration

Quality and in vitro fertilizing ability of frozen-thawed cat semen collected by urethral catheterization (CT) or electroejaculation (EE) after medetomidine administration were compared. Sperm collection was performed by an urinary tomcat catheter and, 4 days apart, by electroejaculation from each of eight tomcats. Results showed that semen collected by CT was characterized by lower volume (10.5 ± 5.3 μL, P < 0.05), higher sperm concentration (1868.4 ± 999.8 × 10 6/mL, P < 0.05) and lower pH (7.0 ± 0.4, P < 0.05) than that collected by EE (67.1 ± 25.9 μL, 542.9 ± 577.9 × 10 6/mL, and 7.9 ± 0.4, respectively). Spermatozoa characteristics after thawing at 0, 3 and 6 h did not differ between the two methods of collection. Also cleavage rate and embryo production from oocytes fertilized with frozen-thawed spermatozoa collected by CT or EE showed no significant differences ( P > 0.05). In conclusion, the results obtained in the present study indicate that good quality freezable semen can be collected from cats by urethral catheterization after medetomidine administration. This new method of semen collection appears very useful in practice and, compared with the electroejaculation protocol, permits to obtain semen samples characterized by a higher concentration of spermatozoa, lower total volume and lower pH.

The sex-ratio of singleton offspring in assisted-conception pregnancies

To evaluate the effect of treatment and male factor infertility on the sex ratio of singleton births from assisted reproductive technology (ART). Retrospective analysis of singleton live births in the 2005 SART database, including fresh and frozen cycles, autologous and donor oocytes. The study population included 19,048 singleton pregnancies. The data were grouped into four cohorts defined by male factor infertility and either no use of ICSI or ICSI in some or all of the eggs. Factors studied included: maternal age, gravidity, diagnosis, oocyte and semen sources, semen collection method, assisted hatching, and number of oocytes or embryos transferred. Logistic regression was used to model the probability (odds ratio, OR, and 95% confidence interval, CI) of male infant overall, and within three maternal age groups (<35 years, 35–40 years, and >40 years). In the general population in the United States in 2004, 51.2% of live births were male. In the study population, the percentage of males was 49.9%, including 51.7% for births without ICSI and 48.9% with ICSI. The major factors influencing the sex ratio were use of ICSI, presence of male factor infertility and their interaction. The sex ratios for these factors are presented in the table below. Although all factors in the original model differed significantly between the four cohorts defined by ICSI and male factor infertility, none of them affected the sex ratio. The pattern of sex ratio across the four cohorts did not change after stratification by maternal age. TableNo ICSI No Male FactorNo ICSI Male FactorICSI No Male FactorICSI Male FactorN5,9915256,1396,393% Male (all)52.147.247.850.0OR (95% CI)1.00 (Reference)0.82 (0.69–0.98)0.84 (0.78–0.91)0.92 (0.86–0.98)% Male (Mothers <35 yrs)53.147.848.149.5OR (95% CI)1.00 (Reference)0.80 (0.61–1.05)0.82 (0.73–0.92)0.87 (0.78–0.96)% Male (Mothers 35–40 yrs)51.146.148.449.8OR (95% CI)1.00 (Reference)0.83 (0.61–1.14)0.90 (0.79–1.02)0.95 (0.84–1.08)% Male (Mothers >40 yrs)51.645.146.350.8OR (95% CI)1.00 (Reference)0.76 (0.50–1.16)0.82 (0.69–0.96)0.98 (0.81–1.19) Open table in a new tab Male factor infertility and the use of ICSI may reduce the chances of having a male child, although the mechanisms remain unclear.

Trehalose enhanced the freezability of Poodle dog sperm collected by an artificial vagina (AV)

In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen–thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm.