Production and storage of goat semen for artificial insemination

Environmental influences on reproduction and semen production in the buck, the problem of interaction between seminal plasma and egg yolk or milk constituents in diluent, liquid storage and processing of semen for freezing are discussed. A review is given on the use of frozen-thawed semen for artificial insemination (AI) in spontaneous and induced oestrus and factors influencing the fertility.     

Collection of semen and artificial insemination of alpacas

Semen collection and artificial insemination have not yet been fully developed in the alpaca. Thus, we collected semen from 7 males using a modified artificial vagina placed inside a dummy. Forty adult female alpacas, previously induced to ovulate with hCG, were artificially inseminated with fresh undiluted semen by laparoscopy or by cervix. The Chisquare test was used to determine differences in the fertility rate of the 2 insemination methods. The mean duration of copulation, semen volume, sperm concentration and the percentages of live spermatozoa and normal spermatozoa were 21.6 min, 1.9 ml. 147,500/mm(3), 69.6% and 75.9%, respectively. There were 6.7% abnormal heads, 12.3% abnormal tails and 3.8% cytoplasmic droplets. The consistency of semen was viscous and formed a coagulum. The pH was 7.2, and the semen was milky white in color. The duration of copulation was comparable to natural copulation, and semen characteristics reflected those of the natural ejaculate. The percentage of pregnancy was 68%, with no differences due to method of semen deposition (laparoscopy, 67%; cervix, 73%).     

Storage of semen and artificial insemination in deer

Methods of collection and freezing of semen of some deer species and aspects of controlled reproduction associated with the use of frozen-thawed semen by artificial insemination (AI) are discussed.     

Hygienic aspects of storage and use of semen for artificial insemination

The artificial insemination (AI) industry has developed over the last 50 years to the extent that it is used in almost every country in the world. One of the main factors contributing to its success is the confidence of the farmers that germplasm is not associated with pathogens, so that AI can be performed without risks. This has been achieved as a result of a considerable amount of research based on sound scientific data that has identified the major risk pathogens. A summary of these studies, given in this section, shows that despite the large number of agents that could be transmitted via the semen, there are cost-effective means to prevent such hazards. One of the basic rules is that the males should be housed in strictly protected semen collection centres (SCCs). Such centres should be approved by the veterinary authorities based upon specific criteria, which include special housing and operating specifications. This also includes specific means of monitoring the health of individual males through regular clinical examinations, assessment of semen and testings for various diseases. Two new challenges can now be identified, one relevant to so-called emerging diseases the impact of which on the status of the semen donors should always be assessed, and the second, relates to endangered genetic resources which may become extinct without active conservation programmes. The experience gained by the AI industry over the last 50 years should help to solve those problems. Currently, the use of semen derived from approved SCCs warrants their disease-free status.     

Prevalence of chlamydiae in boars and semen used for artificial insemination

Although there are indications for venereal transmission of chlamydiae in pigs, direct diagnostic evidence on the presence of these bacteria in boars and boar semen in particular is still incomplete. We investigated boars from two studs (A, B) in semen (A: n = 174; B: n = 100) and faeces (A: n = 174; B: n = 24) for chlamydiae using ompA-PCR and partial ompA gene sequencing. Additionally, blood serum was examined for chlamydial antibodies using an indirect ELISA (A: n = 171; B: n = 62). Chlamydiae were found in 9 (5.2%) and 24 (24.0%) semen specimens, and in 71 (40.1%) and 2 (8.3%) faecal samples from boars of stud A and B, respectively. Regarding individual chlamydial species, Chlamydophila psittaci and Chlamydia suis were identified most frequently, with the former predominating in semen (in 23 out of 33 positive samples) and the latter in faeces (68/73). In contrast, Chlamydophila pecorum was found only sporadically. Chlamydial antibodies were detected in 80 (46.8%) and 6 (9.7%) boars of stud A and B, respectively. No correlation was observed between the data from serology and PCR of semen or faeces in either of the studs. In conclusion, detection of chlamydiae in semen of boars suggests a potential for venereal transmission. Whether the high overall prevalence of chlamydial infections reflects a general situation in boars needs to be investigated. Serological testing failed to identify boars shedding chlamydiae in their semen.     

The relationship between rabbit semen characteristics and reproductive performance after artificial insemination

The relationships between several rabbit buck semen traits, concerning either the ejaculate or the dose inseminated (volume, mass motility, pH, percentage of motile sperm (PMS), concentration, number of total and motile sperm per ejaculate and per insemination dose) and the reproductive performance of does was investigated in 839 inseminations involving 54 bucks and 111 does. Four genetic types were involved: INRA1601 strain (A), INRA2066 strain (B) and the two reciprocal crossbreds (AB and BA). The mating design was A x A, B x B, (AB or BA) x (AB or BA). Semen was diluted (1:9) and a constant volume of 0.5 ml was inseminated 2-4h after collection. Therefore, the total number of spermatozoa per dose was variable and proportional to the initial concentration. Mass motility significantly influenced the kindling rate. Taken separately, volume, PMS and concentration did not influence the kindling rate but their product, the number of motile sperm per ejaculate, did. Litter size (total born) was significantly influenced by concentration and all variables depending on it, particularly the number of total and motile sperm per dose. However, reproductive performances were predominantly influenced by the physiological status of the does at insemination (lactation stage and receptivity).     

Seasonal effects of semen collection and artificial insemination on dairy cow conception

The effects of four seasons of semen collection and of artificial insemination on conception in dairy cows were studied. The solstices and equinoxes (December, March, June and September) defined the beginning and/or end of each season. Semen was collected from 973 progeny-test bulls over 8 years at the two Norwegian AI stations at 60.8 degrees N and 63.4 degrees N where artificial light was used to provide a minimum photoperiod of 10 h/day. The effect of using semen of elite bulls during progeny testing and after selection as elite sires also was investigated. Norwegian Red (NRF) cows were inseminated over a 7-year period using progeny test semen and over the last 4 years of the same period using the semen of the elite sires. The probability of conception to only first inseminations for cows up to, and including, the fifth lactation was assessed by 56-day non-return rate (56d NRR) and calving rate. Two data sets were analysed which excluded cows culled within 270 days of AI or included such cows as non-calving. The reasons for culling were categorised as those for fertility problems or all other reasons. Semen was used for AI irrespective of the season in which it had been collected. Season of semen collection did not affect 56d NRR but calving rate was significantly higher (by 0.5-0.8%, approximately; P < 0.01) for semen collected in the December-March period, when photoperiod was increasing, than at other times of the year. The season in which AI was performed showed a peak of 56d NRR in spring for heifers (P < 0.01) and in summer for parous animals (P < 0.01). For calving rate, however, no seasonal peak was found in heifers, whereas pluriparous cows had much higher calving rates in summer and autumn/early winter than late winter and spring (P < 0.01). Semen of elite sires resulted in higher calving rates by 0.5 (NS) to 1.9% (P < 0.01) when used after selection than when used during progeny testing. The difference between the calving rate achieved when the semen from elite sires was used during progeny testing and after selection indicates that farmers select different classes of cows for submission to AI by progeny test bulls and sires. The 56d NRR was not as good as calving rate for assessing seasonal and other effects on conception rates.     

The potential transmission of infectious agents by semen packaging during storage for artificial insemination

Plastic straws, of a type widely used for semen cryopreservation, sealed using three different methods, (PVA powder, plastic spheres and plasticine modelling clay) were tested for leakage of low molecular weight dye (methylene blue), bacteria (Escherichia coli) and virus (Newcastle disease virus). Leakage was found to be dependent on the method used to fill the straws. Straws filled using a traditional ‘dip and wipe’ method and sealed with PVA powder demonstrated a significant degree of methylene blue leakage (0.0269% of the total straw contents) probably associated with contamination of the powder sealing plug. Straws filled using an aseptic filling technique showed no detectable leakage of any agent with any of the sealing methods. This study highlights the need to establish good-practice guidelines for the packaging of semen collected for freezing and future AI from non-domestic livestock where disease-free status cannot be guaranteed and unsophisticated technology is used.     

Fertility of ram semen frozen in Bioexcell and used for cervical artificial insemination

The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.     

Development of a new transcervical artificial insemination method for sheep: effects of a new transcervical artificial insemination catheter and traversing the cervix on semen quality and fertility

The difficulty of traversing the cervix severely limits transcervical artificial insemination (TC AI) in sheep. Cervical trauma and poorly designed instruments can reduce fertility after AI. To overcome problems associated with TC AI, we developed a new TC AI catheter. Three bench experiments were conducted to determine the effects of the new TC AI catheter on semen quality independent of the effects of moving the catheter through the cervix. In each of the three bench experiments, the standard laparoscopic instrument for intrauterine AI in sheep was used as the control for the TC AI catheter. In Experiment 1, the total volume of semen extender expelled and void volumes for both types of AI instrument (TC versus laparoscopic) were determined. In Experiment 2, the effects of each type of AI instrument (TC versus laparoscopic) on semen quality, estimated as percentage motility and percentage forward progressive motility, of frozen-thawed semen was determined. In Experiment 3, the effects of both types of AI instrument (TC versus laparoscopic) on number of spermatozoa expelled was determined. The type of AI instrument affected neither semen quality nor the number of spermatozoa expelled. However, void volume differed (P < 0.01) between the two instruments. After differences in void volume were taken into account, an in vivo experiment was conducted to determine whether using our new TC AI catheter for TC or surgical intrauterine AI affected fertilization and pregnancy rates. For this, ewes were assigned to one of three treatments: (1) TC AI using the new TC AI catheter + sham AI via laparotomy (n = 9); (2) sham TC AI + AI via laparotomy using a laparoscopic AI instrument (n = 8); and (3) sham TC AI + AI via laparotomy using the new TC Al catheter (n = 10). To synchronize estrus, progestogenated pessaries were inserted and left in place for 12 days. On Day 5 after pessary insertion, PGF2alpha (15 mg) was given i.m. At pessary removal, 400 IU of eCG were administered i.m. Ewes were inseminated 48-52 h after pessary removal using fresh diluted semen (200 x 10(6) to 350 x 10(6) spermatozoa per 0.2 ml) pooled from the same four rams each day during the experiment. At 72 h after AI, uteri were collected postmortem and flushed. Oocytes and embryos were recovered and evaluated. Treatments did not affect (P > 0.01) ovum and embryo recovery rate (mean = 87.3%), fertilization rate (59.3%), or Day 3 pregnancy rate (mean = 66.6%). We conclude from these data that the use of our new TC AI catheter for TC AI or intrauterine AI should not impair the success of AI in sheep.     

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.     

Radiographic contrast medium for uterine insemination in the bitch, and its effect upon the quality and fertility of fresh dog semen

Four ejaculates were collected from each of 6 adult male beagle dogs. The second fraction was divided into 3 aliquants which were then diluted with physiological saline, an isoosmolar solution of sodium/meglumine diatroate, and an iso-osmolar solution of iohexol. The diluted samples were incubated at 39 degrees C and evaluated at 0, 60, 90, 120, 240 and 360 min after dilution. A variety of assessments was made, including, spermatozoal motility, spermatozoal morphology, and acrosorne status. The practicality of using 1.0 ml of iso-osmolar contrast medium combined with radiographic examination was evaluated as a method of confirming accurate placement of a transcervical uterine catheter by injecting contrast after positioning the catheter in 4 beagle bitches. The effect of the procedure on fertility was assessed using 5 greyhound bitches which were inseminated with fresh semen and in which pregnancy was monitored using diagnostic B-mode ultrasound imaging. There was no significant difference between physiological saline and the sodium/meglumine diatroate solution upon semen quality, while the iohexol solution produced a significant reduction in spermatozoal motility and morphology. No adverse clinical effects were observed when contrast medium was administered into the uterus to either group of bitches. A subjective assessment of radiographic quality showed that the sodium/meglumine diatroate solution, which contained twice the iodine concentration of the iohexol solution, produced significantly greater radiopacity and was radiographically more useful than the iohexol solution. The sodium/meglurnine diatroate solution had no adverse effect upon the fertility of dog semen, and all bitches that were inseminated with this technique conceived and maintained the pregnancy to term. Litter size was considered to be normal for the breed. Small volumes of an iso-osmolar solution of sodium/meglumine diatroate may be useful for ensuring correct placement of transcervical catheters prior to artificial insemination in the bitch.     

Artificial insemination of sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen.

Results from an artificial insemination (AI) experiment revealing the effect of semen dilutents containing egg yolk on the fertility of ram semen are presented. Ram semen was diluted 30-fold in buffered glucose-saline solution containing . 375, 1.5, or 6% v/v egg yolk and a portion of each was used soon for the AI of ewes or was incubated at 35 degrees C for 1 hour prior to AI. Some of the semen collection was used undiluted for AI of 10(8) spermatozoa/dose. All diluted samples were reconcentrated by centrifugation so that each dose was 10(8) spermatozoa in a volume of 100 mcl. 1146 ewes were inseminated. Fertility was assessed from 28 to 45 day nonreturns to estrus and nonreturn rates (NRRs) were expressed as percentages for the various treatments. Undiluted semen (controls) revealed 69%, semen used soon after dilution, .375% yolk in dilutent 58%, 1.5% yolk 50%, 6% yolk 42%; diluted semen incubated for 1 hour before use, .375% yolk 49%, 1.5% yolk 51%, and 6% yolk 39%. NRR was significantly depressed by dilution (p less than .001) and by increasing amounts of egg yolk (p less than .01) in the dilutent. Incubation of diluted semen before AI caused a small fall in NRR.     

Efficiency of photosynthesis in continuous and pulsed light emitting diode irradiation

The light utilization efficiency and relative photon requirement of photosynthesis in pulsed and continuous light from light emitting diodes (LEDs) has been measured. First, we chacterized the photon requirement of photosynthesis from light of LEDs that differ in spectral quality. A photon requirement of 10.3±0.4 was measured using light from a 658 nm peak wavelength (22 nm half band width) LED over the range of 0–50 mol photons m^–2 s^–1 in 2 kPa O₂ in leaves of tomato (Lycopersicon esculentum Mill., cv. VF36). Because the conversion of electrical power to photons increased with wavelength, LED lamps with peak photon output of 668 nm were most efficient for converting electricity to photosynthetically fixed carbon. The effect of pulsed irradiation on photosynthesis was then measured. When all of the light to make the equivalent of 50 mol photons m^–2 s^–1 was provided during 1.5 s pulses of 5000 mol photons m^–2 s^–1 followed by 148.5 s dark periods, photosynthesis was the same as in continuous 50 mol photons m^–2 s^–1. When the pulse light and dark periods were lengthened to 200 s and 19.8 ms, respectively, photosynthesis was reduced, although the averaged photon flux density was unchanged. Under these conditions, the light pulses delivered 10¹⁷ photons m^–2, which we calculate to be equivalent to the capacitance of PS I or PS II. Data support the theory that photons in pulses of 100 s or shorter are absorbed and stored in the reaction centers to be used in electron transport during the dark period. When light/dark pulses were lengthened to 2 ms light and 198 ms dark, net photosynthesis was reduced to half of that measured in continuous light. Pigments of the xanthophyll cycle were not affected by any of these pulsed light treatments even though zeaxanthin formation occurred when leaves were forced to dissipate an equal amount of continuous light.Abbreviations CWF cool white fluorescent - EPS xanthophyll epoxidation state - LED light emitting diode - LUE light utilization efficiency - PFD photon flux density - PR photon requirement (for CO₂ fixation) - PS II primary donor in Photosystem II - RPR relative photon requirement     

Prevalence of PCV2 in Austrian and German boars and semen used for artificial insemination

Since epidemiologically-based science on PCV2 in porcine semen is patchy, we investigated 806 Austrian (A) and German (G) AI boars from five studs, and boars from Austrian farms used for on-farm semen collection, for the presence for IgG/IgM in blood by ELISA (n=754) as well as for PCV2 DNA in semen (n=472) and if positive, also in blood of a few boars by nested PCR and sequencing. A total of 420 boars were tested for both PCV2 in semen and antibodies in blood. Boars were aged between 8 and 82 months at sampling. None of the boars tested positive for IgM but 60.1% did for IgG. PCV2 DNA was detected in 86 (18.2%) semen samples. Minor differences were found between boar populations with respect to the number of antibody positive boars and no differences for DNA in semen. Phylogenetic analysis of 28 sequences revealed a genetic diversity of PCV2 in semen within and between boar populations, with sequences belonging to both PCV2 genotypes 1 and 2. Mean nucleotide sequence identity was 95.7%, with maximum pairwise difference of 8.8%. Boars < or =16 months were tested more frequently positive for IgG (P<0.001) and for PCV2 DNA in semen (P<0.05) than older boars. Of 80 boars tested positive in semen, 34 (42.5%) were antibody negative. A total of 58 semen positive boars with (n=33) and without (n=25) IgG were all tested negative for PCV2 DNA in serum. In conclusion, this study demonstrated the ubiquity of PCV2 in the Austrian and German boar population. Genetically diverse PCV2 can be encountered in boar semen. Shedder boars cannot be detected on the basis of serology. There is an apparent possibility of PCV2 being transmitted through semen.     

LED光照对棉铃虫成虫明适应状态和交尾的影响

室内条件下研究了505、540nm和590nm 3种波长LED(light emitting diode)光照对棉铃虫成虫明适应状态和交尾的影响.结果表明,夜间给予3种波长的LED光的持续光照和间歇光照均能有效地保持棉铃虫成虫的明适应状态,明适应保持率在75%～100%;590nm的LED光照能够使棉铃虫成虫的交尾率由对照组的89.1%降低至52.5%,同时明显减少其交尾次数.本试验的结果还表明,505nm和590nm的LED光照能够改变棉铃虫成虫的产卵动态,使平均产卵期延长2d左右,同时也能显著地降低棉铃虫卵的孵化率,但对棉铃虫产卵量的影响不显著.