Adjusted method of penis fixation during boar semi-automatic semen collection aiming to reduce bacterial contamination

Semen collection has an essential role in the initial bacterial load in boar ejaculates and extended semen. The study aimed to explore the efficacy of an adjusted penis fixation in a semi-automatic collection system on reducing bacterial contamination of ejaculates in two-boar studs with different scenarios. Historically, stud A had low levels of bacterial load in raw semen, while stud B had a high level of contamination. A total of 56 mature boars had their semen collected using two methods of penis fixation: (a) Traditional: The penis was fixed directly with the artificial cervix and transferred to the adjustable clamp; (b) Adjusted: The fixation was performed with one gloved-hand, and after exteriorization, the penis was gripped using the artificial cervix with the other gloved-hand and transferred to the adjustable clamp. The bacterial load (p = .0045) and the occurrence of ejaculates >231 CFU/ml (p = .0101) were reduced in the Adjusted compared to the Traditional method. Bacterial load was reduced when using the Adjusted method in stud B (p = .0011), which showed a greater occurrence of critical factors for bacterial contamination (p ≤ .0034). The Adjusted method reduced the occurrence of ejaculates >231 CFU/ml when the preputial ostium was dirty (p = .016) and the duration of semen collection was >7 min (p = .022) compared to the Traditional method. In conclusion, the Adjusted penis fixation was efficient in reducing bacterial load of ejaculates, mainly in boar stud B, which had high contamination challenges.     

Application of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Identification of Stallion Semen Bacterial Contamination

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the cutting-edge methods currently applied in medical bacteriologic diagnostics. The aim of the study was to prove the possibility of applying MALDI-TOF MS to identify bacterial contamination in the ejaculate of stud stallions, which may cause infections to reproductive organs of mares following artificial insemination with cooled semen. A partial aim was to determine changes in the total count of microorganisms in long-term storage of ejaculate after its treatment with gentamicin and also without antimicrobial medication. Aerobic cultivation on Columbia agar was used to examine 26 semen samples from 13 horses; 31 different species of bacteria were isolated, which were identified by MALDI-TOF MS. The most frequently detected species came from Aerococcaceae, Staphylococcaceae, and Micrococcaceae families. The results of our work confirm that MALDI-TOF MS is a quick alternative method for identifying bacterial species that may contaminate stallion semen.     

Bacteriological findings in different fractions of canine ejaculates showing normospermia,teratozoospermia or azoospermia

Objective There is little available information on the distinction between physiological and pathological bacterial flora in dog semen, or in fractions of the canine ejaculate. In order to improve the diagnostic basis, the present study describes the bacteriological findings in canine semen samples. Design and procedure The ejaculates were assigned to three groups: Group A, normospermia (n = 30); Group B, teratozoospermia (n = 33); Group C, azoospermia (n = 29). Results Our data show that irrespective of semen quality, bacterial contamination and stronger bacterial growth is more common in the pre‐sperm fraction than in the other two fractions. The number of bacteria and bacterial species did not vary between the three fractions or the three groups of dogs. Bacteria regarded as potentially pathogenic (β‐haemolytic Streptococci, Escherichia coli variation haemolytica) could be isolated in every group, with β‐haemolytic Streptococci tending to occur more frequently in all ejaculate fractions from dogs with teratozoospermia. Conclusion These data emphasise that the first fraction is more contaminated with bacteria than the second and third fractions which may be due to bacteria ascending from the urethra. The use of a fractionated collection of canine ejaculates seems to be beneficial to reduce or prevent bacterial contaminations.     

Semen collection and evaluation

Conformation showing and a variety of dog sports are increasingly popular. Propagation of an excellent dog requires normal fertility. Semen collection and evaluation are essential skills for small animal practitioners who work with breeders and dog-sport enthusiasts. Semen collection can be performed on most dogs in the clinic setting with minimal standard equipment. Preparation of the collection room and presence of a teaser bitch increase the success of sample collection. Semen evaluation consists of several components. First, gross characteristics and sample volume are recorded. Percentage and quality of motility are evaluated immediately. The concentration of spermatozoa in the sample is determined and multiplied by the total sample volume to determine the total number of sperm in the ejaculate. Morphology of > or = 100 spermatozoa is evaluated and recorded to determine the percent normal spermatozoa Total normal sperm per ejaculate is then calculated. Additional tests are often performed. These include cytology of the second and third fractions, culture of the semen, and measurement of semen alkaline phosphatase. Knowledge of normal parameters for canine semen enables the practitioner to evaluate the results of these tests with confidence.     

Antibiotics for the preservation of koala (Phascolarctos cinereus) semen

Aim To determine the normal microbial flora of the koala ejaculate and prepuce in order to select appropriate antibiotics for addition into diluents designed for the preservation of semen.
Procedure Bacteriological samples of the koala prepuce (n = 12) and ejaculate (n = 20) were submitted for microbial culture and sensitivity testing. Microbial flora of ejaculates collected by electroejaculation and artificial vagina were compared. The effects of varying concentrations of penicillin G and gentamicin on sperm motility and on the growth of bacteria in diluted semen stored at room temperature and 16°C over a 24 h period were investigated.
Results A range of bacteria was isolated from the koala prepuce and ejaculate. The predominant organisms in semen collected by electroejaculation and artificial vagina were Corynebacterium spp, none of which could be assigned to any recognised species. The addition of penicillin G and gentamicin to a PBS-based diluent at dose rates of 1000 to 2000 IU/mL and 100 to 200 m g/mL respectively, resulted in no adverse effect on sperm motility over a 24 h incubation period. Penicillin G (1000 IU/mL) and gentamicin (100 m g/mL) prevented growth of bacterial contaminants in diluted koala semen.
Conclusion By controlling the growth of bacteria in extended koala semen, penicillin G and gentamicin are likely to lengthen the period by which spermatozoa can be stored at 16°C and reduce the possibility of disease transmission during artificial insemination procedures.     

Preservation of boar semen: An update

In the pork industry, artificial insemination and the storage of boar semen in liquid at 17°C are routinely applied to optimize the ejaculate and bring about rapid genetic changes that are reflected in the animal protein. Although the results are satisfactory, they are below what occurs with natural mating. It is currently possible to preserve boar semen with storage at 17°C and slow freezing, since to date there is only one study on vitrification, with negative results applicable only in the case of implementing an intracytoplasmic sperm injection. In both methods and due to the sensitivity of boar sperm to osmotic and temperature changes, there is a loss in the quality of the initial sample; however, slow freezing in boar semen has greater deleterious effects on the sample that are reflected in the pregnancy rates and number of live births. Therefore, only 1% of all inseminations are done with frozen semen. The aim of this review is to provide advances and results of studies conducted on the preservation of boar semen, delving more deeply into the critical points that each of the preservation techniques presents, including bacterial contamination, extender components, temperature, ice nucleation, use of additives in extenders and the main deleterious effects on sperm quality.     

Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen

Bacteria contaminate semen during collection and handling. The objective of this study was to identify the bacteria in pony stallion semen, the effects of antibiotics included in commercial semen extenders (lincomycin and spectinomycin) and the effect of modified single layer centrifugation (MSLC), on bacterial load. Ejaculates from six pony stallions, 3 ejaculates per animal, were extended in EquiPlus extender either with or without antibiotics. Aliquots were processed by MSLC to form four treatment groups: control and MSLC with antibiotics (CA and SA, respectively) and control and MSLC without antibiotics (CW and SW, respectively). Bacteriological examinations were carried out within 2 hr. Thirty‐one species of bacteria were isolated from one or more ejaculates, with Corynebacterium spp. being the most frequently detected. Corynebacterium spp. were present in all ejaculates. The MSLC resulted in a significantly lower total bacterial count than controls (CA vs. SA, p < 0.001; CW vs. SW, p < 0.0001).     

Evaluation of surveillance protocols for detecting porcine reproductive and respiratory syndrome virus infection in boar studs by simulation modeling.

Because porcine reproductive and respiratory syndrome virus (PRRSV) can be transmitted through semen, PRRSV-free boar studs need to be routinely monitored to rapidly detect any potential PRRSV introduction. However, current protocols for monitoring PRRSV in boar studs are diverse, sometimes very costly, and their effectiveness has not been quantified. The objective of this study was to evaluate the ability of different monitoring protocols to detect PRRSV introduction into a negative boar stud by using a simulation modeling approach. A stochastic transmission model was constructed to simulate the spread of PRRSV in a typical negative boar stud in the USA (herd size of 200 boars, 60% annual replacement) and the performance of monitoring protocols by using different sample sizes (10, 30, and 60 samples), sampling frequency (3 times a week, weekly, and biweekly), and diagnostic procedures (PCR on semen, PCR on serum, ELISA on serum, and both PCR and ELISA on serum). The monitoring protocols were evaluated in terms of the time from PRRSV introduction into the boar stud to PRRSV detection. Protocols that used PCR on serum detected the PRRSV introduction earlier than protocols that used PCR on semen, and these were earlier than those that used ELISA on serum. The most intensive protocol evaluated (testing 60 boars 3 times a week by PCR on serum) would need 13 days to detect 95% of the PRRSV introductions. These results support field observations, suggesting that an intensive monitoring protocol needs to be in place in a boar stud to quickly detect a PRRSV introduction.     

Characterization of semen collected by pharmacologically induced ejaculation from a stallion with seminal vesiculitis

The present study compared the quality of sperm collected by artificial vagina or pharmacologically induced ejaculation from a 10-year-old thoroughbred stallion with seminal vesiculitis. The pharmacological protocol involved intravenous administration of detomidine (0.01 mg/kg) and oxytocin (20 IU) and successfully induced ejaculation in all attempts of semen collection. Sperm motility, plasma membrane and acrosome integrity (PMAI), reactive oxygen species (ROS) levels, polymorphonuclear neutrophil (PMN) percentage, and bacterial profiles of fresh and cooled semen (5°C for 24 hr) were evaluated. Semen obtained by the pharmacological method presented reduced seminal volume, decreased PMN percentage and superior sperm motility in cooled samples. Moreover, higher PMAI and lower ROS levels were observed in semen collected by the pharmacological method. Therefore, pharmacologically induced ejaculation is an alternative to obtain semen with minimal contamination and with sperm of superior quality and longevity from stallions with seminal vesiculitis.     

Bacterial shell contamination in the egg collection chains of different housing systems for laying hens

The bacterial eggshell contamination of eating eggs in different commercial housing systems; two conventional cages, one organic aviary system and one barn production, were compared. The total counts of aerobic bacteria and the total counts of Gram-negative bacteria on the shell were used to detect key points where contamination occurred and to study the progress of contamination in the egg collection and transportation chains. The key points in the chain were those where eggs accumulated on a short conveyor belt, initial shell contamination in the alternative housing systems and extra nest-boxes placed on the ground. The high bacterial load of floor eggs (>6.3 log CFU total aerobic flora/eggshell) explains why they cannot be used for eating. On average higher initial shell contamination with total counts of aerobic bacteria was found for eggs from the alternative housing systems compared to the conventional systems; respectively 5.46 compared to 5.08 log CFU/eggshell. However, initial contamination with total counts of Gram-negative bacteria on the shells was less in the alternative systems: 3.31 compared to 3.85 log CFU/shell. Initial bacterial shell contamination tended to correlate positively with the concentration of bacteria in the air of the poultry houses. Storing shell eggs, whether temporarily refrigerated or not, for 9 d or more, resulted in a decrease in bacterial eggshell contamination for both bacterial variables.     

The current position of A.I. in horse breeding.

This short review article describes the various techniques currently available for artificial insemination in the horse. The collection and use of raw and extended semen is discussed together with the more recent developments in freezing semen. The expected conception rates with both fresh and frozen semen are quoted. The possible benefits in disease control and stud management are discussed, as well as the difficulties in controlling the use of A.I. from the Breed Registration Authorities point of view.     

Swine diseases transmissible with artificial insemination

The transport of fresh and frozen semen to be used for artificial insemination creates a mode of disease transmission between farms. Normally, semen contains a number of nonpathogenic bacterial contaminants; however, excessive bacterial contamination can result in infertile matings. Contamination with a known pathogen, eg, Brucella suis, could initiate a serious outbreak of disease in a recipient herd. Methods to minimize bacterial contamination of semen include sanitary collecting and processing of semen, isolation of boards from certain pathogens, and the addition of appropriate broad spectrum or combination antibiotics to the semen. Mycoplasmas also have been isolated from semen, although transmission by this route is unlikely. The addition of an appropriate antimycoplasmal antibiotic to semen may be warranted in some situations. Numerous viruses have been detected in semen. Their exclusion from semen is especially critical because of their ability to survive in frozen semen. These viruses include pseudorabies virus, porcine parvovirus, enterovirus, adenovirus, vesicular disease virus, and African swine fever virus. The likelihood of disease transmission is greater with the introduction of a boar into a herd than through the use of fresh or frozen semen. We believe that artificial insemination allows for the introduction of new genetics into a breeding program, with minimal risk of disease transmission.     

Evaluation of stallion semen.

This article outlines a basic method for conducting a stallion semen evaluation. After the removal of the gel fraction of the ejaculate, semen gel-free volume is determined, and any abnormality in appearance is noted. Concentration of sperm cells in semen can be determined with the use of either a hemacytometer or spectrophotometer after appropriate dilution of raw semen. The percentage of progressively motile sperm is evaluated promptly after collection of semen with the use of a phase-contrast microscope. The total numbers of sperm and progressively motile sperm in the ejaculate are calculated. The determination of seminal pH and the classification of sperm morphologic features are additional seminal characteristics evaluated during a semen evaluation. Sperm motion characteristics can be further evaluated with the use of computerized sperm image analysis systems and may add additional information concerning the quality of ejaculated sperm. Unfortunately, no single seminal characteristic has in itself been shown to be highly correlated with fertility, although various seminal characteristics are known to affect fertility. Therefore, to properly interpret the fertility of a semen sample, a complete and thorough semen evaluation must be performed.     

Genetic Parameters for Semen Production Traits in Austrian Dual-Purpose Simmental Bulls

Genetic parameters were estimated for semen production traits collected in an Austrian AI centre in the years 2000-2004. In total, 12,746 ejaculates from 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls were examined considering different effects on ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. The model for genetic parameter estimation included the fixed effects age of bull, collection interval, number of collections on collection day, bull handler, semen collector, year and month of collection, a random additive genetic component and a permanent environmental effect. Correlations between estimated breeding values for semen traits and male fertility from the routine evaluation were calculated. The fertility trait considered in the routine evaluation is non-return rate 90 for the first insemination. All semen production traits were moderately heritable. Heritabilities for volume, concentration, percentage of viable spermatozoa, total number of spermatozoa and motility were 0.18, 0.14, 0.10, 0.22 and 0.04, respectively. Correlations between breeding values for semen quality traits and routinely estimated breeding values for male fertility were low and ranged from 0.08 to 0.17 indicating that semen production traits are rather poor predictors of male fertility.     

广州地区娟姗种公牛精液品质随月份的变化研究

选择在广州地区亚热带气候条件下2岁～6岁的娟姗公牛5头,观察其精液品质(采精量、原精密度、原精活力以及细管精液产量)在全年不同月份的变化情况.结果表明:娟姗公牛的每次采精量和细管精液产量在气温较高的7～9月份会因为采精频率的减少而增加,10月份会因为周采精频率增加而使每次采精量和细管精液产量有明显减少;娟姗公牛的原精密度在气温适宜的2月份和采精频率较低的8月份会出现两个高峰值;娟姗公牛的原精活力在气温较高的月份会出现明显的下降趋势,2月份气温适宜时原精活力最好.     

Effect of collection frequency on quantitative and qualitative characteristics of pigeon (Columba livia) semen

The aim was to estimate the optimal frequency of semen collection from pigeons in relation to ejaculate volume, sperm concentration, total spermatozoa in ejaculate and percentage of live morphologically normal cells. The study was carried out on 455 ejaculates collected from two groups of pigeons, each of 10 males (group I: meat-type breed; group II: fancy pigeon). The birds were selected and kept individually in cages under a natural photoperiod. A two-person technique was used for semen collection (lumbo-sacral and cloacal region massage). Semen was collected once, twice or three times per week. Colour, consistency and volume of ejaculates were evaluated macroscopically immediately after collection. Sperm concentration and total number of cells in the ejaculate were estimated after dilution with Ringer's solution. A live-dead stain technique (nigrosin-eosin) was used to determine the percentage of live and normal spermatozoa. Semen collected 3x/week was of high quality. The average volume of a single ejaculate was small (21 microl in group I and 19 microl in group II), but sperm concentration was high--1.58 x 10(9)/ml and 1.96 x 10(9)/ml, respectively. The mean number of spermatozoa per ejaculate was 30.48 x 10(6) in group I and 39.49 x 10(6) in group II. An increased percentage of live and normal spermatozoa in semen collected more frequently was also observed. Collecting pigeon semen 3x/week provides spermatozoa in larger amounts and of better quality than less frequent collections (1x/week or 2x/week) and is recommended for obtaining more insemination doses.     

不同采精阶段对后备北极公狐的射精量、精子活力及密度的影响

采用后备北极公狐在配种期内用６个阶段的采精频率检测公狐精液的采出率、射精量、精子活力和密度。精液采出率以３月下旬和４月上旬最高,极显著地高于３月上旬（Ｐ＜００１）;射精量以３月下旬和４月上旬最高,３月上、中旬最低（Ｐ＜００５）;精子活力以４月上旬最高,极显著地高于４月下旬（Ｐ＜００１）,但４月上旬与其他阶段差异不显著（Ｐ＞００５）;精子密度以３月中旬和４月中旬最大,３月下旬最低,二者差异显著（Ｐ＜００５）。结果表明,不同的采精阶段对后备公狐的射精量、精子活力和密度均有一定的影响,尤其对后备公狐发情开始和接近结束阶段的影响更大。     

Prevention of urethral blockage following semen collection in two species of lemur, Varecia variegata variegata and Lemur catta.

Lemurs are a diverse group of primates comprised of five families, all of which are found only on Madagascar and the Comoro Islands. Of the 60 known species, 17 are endangered and 5 of these are considered critically endangered. The effects of inbreeding on population health and viability have been well described; though negative inbreeding effects can be ameliorated through the introduction of new genetic material. Introduction of new individuals into a population can be extremely challenging because of the highly social nature of lemurs. Semen collection in lemur species is notoriously challenging, as the ejaculate forms a coagulum. During normal breeding, the coagulum forms a copulatory plug in the female. However, this coagulum can present a life-threatening situation when retained in the urethra abnormally following electroejaculation. This study investigates the use of ascorbic acid in preventing urethral blockage in two lemur species during semen collection, demonstrates successful collection of semen by electroejaculation from two species of lemur during the breeding season, and discusses removal of urethral plugs subsequent to semen collection. Semen was collected successfully from all animals. Urethral plugs formed during each collection and were abnormally retained in 2/11 collections. Both plugs were successfully and immediately removed with the use of retropulsion through a urethral catheter. Although the results of this study are encouraging, more investigation is required to establish whether or not this procedure can be safely performed in the field.     

Antimicrobial Resistance in Some Gram‐Negative Bacteria Isolated from the Bovine Ejaculate

The bacterial load and degree of antibiotic resistance present in untreated and antibiotic‐treated semen samples were investigated in five bulls standing at a cattle‐breeding centre. Bacterial load was determined by colony counts from semen samples cultured on brain heart infusion and nutrient agar plates. Antibiotic resistance in these bacteria was assessed by measuring the diameter of bacterial growth inhibition zones around discs containing different concentrations of antibiotics. Representative antibiotic‐resistant bacterial isolates were selected for identification. Untreated semen contained few culturable bacteria, and all were completely sensitive to gentamycin, spectinomycin and lincomycin: six of the isolates showed some resistance to tylosin. In semen to which antibiotics had been added as part of the routine production process, two isolates were sensitive to all of the antibiotics tested, and the remainder were resistant to all. Resistant Gram‐negative isolates that were identified included Pseudomonas and Stenotrophomonas spp. both in the class Gammaproteobacteria and a Sphingomonas sp. which is in the class Alphaproteobacteria.     

Genetic parameters for semen production traits in Swiss dairy bulls

Variance components (VC) were estimated for the semen production trait ejaculate volume, sperm concentration and sperm motility in the Swiss cattle breeds Brown Swiss (BS), Original Braunvieh (OB), Holstein (HO), Red‐Factor‐Carrier (RF), Red Holstein (RH), Swiss Fleckvieh (SF) and Simmental (SI). For this purpose, semen production traits from 2,617 bulls with 124,492 records were used. The data were collected in the years 2000–2012. The model for genetic parameter estimation across all breeds included the fixed effects age of bull at collection, year of collection, month of collection, number of collection per bull and day, interval between consecutive collections, semen collector, bull breed as well as a random additive genetic component and a permanent environmental effect. The same model without a fixed breed effect was used to estimate VC and repeatabilities separately for each of the breeds BS, HO, RH, SF and SI. Estimated heritabilities across all breeds were 0.42, 0.25 and 0.09 for ejaculate volume, sperm concentration and sperm motility, respectively. Different heritabilities were estimated for ejaculate volume (0.42; 0.45; 0.49; 0.40; 0.10), sperm concentration (0.34; 0.30; 0.20; 0.07; 0.23) and number of semen portions (0.18; 0.30; 0.04; 0.14; 0.04) in BS, HO, RH, SF and SI breed, respectively. The phenotypic and genetic correlations across all breeds between ejaculate volume and sperm concentration were negative (?0.28; ?0.56). The other correlations across all breeds were positive. The phenotypic and genetic correlations were 0.01 and 0.19 between sperm motility and ejaculate volume, respectively. Between sperm motility and sperm concentration, the phenotypic and genetic correlations were 0.20 and 0.36, respectively. The results are consistent with other analyses and show that genetic improvement through selection is possible in bull semen production traits.