Effect of low and high egg yolk concentrations in the semen extender for goat semen cryopreservation

This research aimed to evaluate two concentrations of egg yolk inclusion rates (20 and 2.5%) in the semen extender of goat semen cryopreserved during two seasons of the year. The study was conducted during a light-induced breeding season (Experiment 1), and during the natural breeding season (Experiment 2), in the southern hemisphere. Four ejaculates from each buck (n = 2) were collected in each experiment. After collection, semen was divided, with each sample being diluted in the semen extender – according to the treatments (T1 – 20% egg yolk or T2 – 2.5% egg yolk, using a glucose–EDTA extender). For T1 treatment in Experiment 2, the semen was also washed before the semen cryopreservation process. The semen samples were frozen, and after thawing evaluated for seminal characteristics i.e. sperm motility, vigor, morphology and membrane integrity. The fertilising capacity of the frozen-thawed semen was evaluated following a single artificial insemination 12 h after the onset of estrus in 50 (Experiment 1) and 60 does (Experiment 2). In Experiments 1 and 2, the mean values for sperm motility and membrane integrity of the frozen-thawed semen did not differ between the T1 and T2 treatments. However, the mean sperm vigor and morphological normal sperm were greater (P < 0.05) in T2 than T1 treatment. The fertility rates recorded did not differ between T1 and T2 treatments in Experiment 1, however, it was greater (P < 0.05) in the T2 than in the T1 treatment, in Experiment 2. According to obtained results, it can be recommended to use a glucose–EDTA extender with a low egg yolk concentration (2.5%) inclusion, for superior fertility results in goats.     

Pregnancy loss in heifers after artificial insemination with frozen-thawed,sex-sorted,re-frozen-thawed dairy bull sperm

The use of sex-sorted sperm by the dairy industry is often limited by the geographical distance between potential sires and the sex-sorting facility. One method that may be used to overcome this limitation is sex-sorting sperm that have been previously frozen, or transported to the sorting facility as cooled liquid semen. In this study the in vivo fertility of frozen-thawed, sex-sorted, re-frozen-thawed (FSF) and cooled, sex-sorted, frozen-thawed (CSF) bull sperm was determined after artificial insemination (AI) of Holstein heifers. Semen from two bulls was frozen in straws, or transported to the sorting facility in an egg yolk diluent at 5 °C over 24 h. Thawed or re-warmed semen was processed through a PureSperm^® density gradient, and sperm were sorted for sex and frozen (2 or 4 × 10⁶ sperm/straw). Synchronised heifers (n = 183) were inseminated with either non-sorted control sperm (Control; 20 × 10⁶ dose) or with FSF or CSF ‘X’ sperm (2 or 4 × 10⁶/dose). Pregnancy rates (detected at 7–9 weeks) after AI with control sperm were higher than with FSF or CSF sperm (57.4 vs. 4.1 and 7.3% respectively; p < 0.001). There was a significant difference between bulls (Bull 1: Control 63.0%, FSF 8.6%, CSF 10.0%; Bull 2: Control 45.5%, FSF 0%, CSF 4.8%; p = 0.001). Five out of six (83.3%) pregnancies produced with sexed sperm were lost after pregnancy diagnosis. The exception was one heifer inseminated with CSF sperm (2 million sperm dose), which produced a heifer calf. In the non-sorted control group, three pregnancies were lost (8.3%) and three stillbirths occurred (8.3%). The low fertility and high rate of pregnancy loss in the sexed groups, in addition to environmental influences, may be attributed to impaired sperm function caused by sex-sorting and re-freezing, leading to poor embryo quality or altered gene expression. More precise timing of insemination and higher sperm doses might improve the fertility of FSF sperm. Moreover, the in vitro function of double-frozen sexed compared with non-sorted sperm requires further investigation.     

Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability

This study compared the field fertility outcomes in frozen–thawed (FT) sperm from boar ejaculates with different freezability (good, GFE/poor, PFE) while testing the reliability of the post-cervical artificial insemination (post-CAI) in FT sperm. The assay was conducted over eight months with 86 weaned sows being inseminated by post-CAI. Every ejaculate in a total of 26 from 15 Piétrain boars was divided into a refrigerated semen portion (FS; control treatment) and a cryopreserved portion (FT sperm), and the ejaculates were in turn classified as GFE or PFE in function of the sperm progressive motility and viability at 240 min post-thaw. As result, one of four possible treatments was randomly given to each sow: FS-GFE, FS-PFE, FT-GFE and FT-PFE. The number of pregnant and farrowing sows in FT-GFE did not significantly differ from those of FS control treatments. Contrarily, the probabilities of pregnancy were two times lower after inseminations with FT-PFE (P < 0.05) compared to FT-GFE, which indicates that ejaculates with high post-thaw sperm progressive motility and viability are more likely to result in pregnancies than those with poor in vitro sperm function. There were no differences in litter size or the risk of backflow among treatments. Further trials are required to determine the optimal volume and concentration of FT sperm in post-CAI to obtain a more reliable method for farmers interested in cryopreserved sperm.     

Application of sperm sorting and associated reproductive technology for wildlife management and conservation

Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.     

Motility and fertility of cryopreserved semen in Persian sturgeon,Acipenser persicus,stored for 30–60 min after thawing

We investigated the effect of storage times of frozen–thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4 °C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.     

Animal board invited review: Germplasm technologies for use with poultry

Over the last century, several reproductive biotechnologies beyond the artificial incubation of eggs were developed to improve poultry breeding stocks and conserve their genetic diversity. These include artificial insemination (AI), semen storage, diploid primordial germ cell (PGC) methodologies, and gonad tissue storage and transplantation. Currently, AI is widely used for selection purposes in the poultry industry, in the breeding of turkeys and guinea fowl, and to solve fertility problems in duck interspecies crosses for the production of mule ducklings. The decline in some wild game species has also raised interest in reproductive technologies as a means of increasing the production of fertile eggs, and ultimately the number of birds that can be raised. AI requires viable sperm to be preserved in vitro for either short (fresh) or longer periods (chilling or freezing). Since spermatozoa are the most easily accessed sex cells, they are the cell type most commonly preserved by genetic resource banks. However, the cryopreservation of sperm only preserves half of the genome, and it cannot preserve the W chromosome. For avian species, the problem of preserving oocytes and zygotes may be solved via the cryopreservation and transplantation of PGCs and gonad tissue. The present review describes all these procedures and discusses how combining these different technologies allows poultry populations to be conserved and even rapidly reconstituted.     

Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.     

Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality

To determine the effects of seminal plasma during and after cyopreservation on post-thaw sperm functions in semen from poor freezability boars, seminal plasma was removed immediately after collection, and sperm was subjected to cooling and freezing. Removal of seminal plasma did not significantly affect post-thaw sperm motility in good freezability boars; however, in boars with poor freezability, it increased post-thaw motility relative to control sperm cooled with seminal plasma (64.5+/-3.4% vs. 30.9+/-3.1%, P<0.01). Freezing sperm without seminal plasma increased both loss of the acrosome cap (37.5+/-1.6% vs. 18.4+/-2.8%, P<0.01) and expression of a 15 kDa tyrosine-phosphorylated protein (capacitation marker) in thawed sperm relative to controls; the addition of 10% (v/v) seminal plasma to the thawing solution significantly suppressed both changes and increased conception rate to AI (70% vs. 9% in the control group, P<0.05). In conclusion, our novel cryopreservation and thawing method increased the success of AI with frozen-thawed porcine semen, particularly from boars with poor post-thaw semen quality.     

Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species

Approximately 503 of the known species of birds are classified as ‘endangered’ or ‘critical’. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific.     

Pregnancy rates in heifers and cows with cryopreserved sexed sperm: Effects of sperm numbers per inseminate, sorting pressure and sperm storage before sorting

Field trials were conducted to increase fertility with AI of flow-sorted, sexed bovine sperm. In the first trial, a novel competitive fertilization approach was used to compare pressures (30 psi vs 50 psi) for sorting sperm. Both X- and Y-sperm were sorted to approximately 95% purity at 30 and at 50 psi; X-50 + Y-30 (and the converse) were mixed in equal numbers for AI of heifers. Fetal sex divulged which treatment produced the pregnancy; 82% of pregnancies resulted from the 30 psi treatment (P < 0.05). Based on a similar approach, a new-pulsed laser did not damage sperm any more than the previous standard continuous wave laser. In a large field trial, sorting sperm at 40 psi increased pregnancy rates in heifers relative to 50 psi (42.3% vs 34.1%, n = 367/group, P < 0.05). Storing sperm for 20 h before sorting at 40 psi decreased pregnancy rates from 42.3% (n = 367) to 36.8% (n = 368; P < 0.05). Breeding heifers with sexed sperm 55-56 h after CIDR removal and PGF_2α resulted in 34% (n = 32) pregnant, compared to 49% (n = 35) with fixed-time insemination 67-68 h after CIDR removal (P > 0.1). Lactating dairy cows pre-screened for normal reproductive tracts when OvSynch injections (GnRH, prostaglandin, GnRH) were initiated, had similar (P > 0.1) pregnancy rates to timed AI, with 10 × 10⁶ sexed sperm (43.9%, n = 57), 2 × 10⁶ sexed sperm (40.5%, n = 57) and 10 × 10⁶ unsexed control sperm (55.6%, n = 58). A final field trial with unselected, lactating dairy cows resulted in similar pregnancy rates for 2 × 10⁶ sexed sperm in 0.25 mL straws (25.0%, n = 708) and 0.5 mL straws (24.4%, n = 776), but lower (P < 0.05) than unsexed control sperm (37.7%, n = 713). Younger cows and those >84 days in milk had the highest pregnancy rates for both sexed and unsexed sperm. These studies improved sperm sexing procedures, and provided insight into appropriate commercial use of sexed sperm.     

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

Double freezing of bovine semen

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3 h incubation at 37 °C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P < 0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P ≤ 0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P < 0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P = 0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.     

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation.Ejaculates collected from three Angora bucks were evaluated and pooled at 37 °C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of CASA motility (63.6 ± 7.0; 63.4 ± 3.1%, respectively), in comparison to the controls (P < 0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P > 0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P < 0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P < 0.05). Malondialdehyde formation was found to be lower (1.8 ± 0.1 nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze-thawing process (P < 0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P > 0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.     

Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyaluronic acid gel

The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre-freeze (“fresh”) and post-thawed (“thawed”) samples from five different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, “Pap”-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.     

Birth of viable puppies derived from breeding cloned female dogs with a cloned male

Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo–matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns.     

Field fertility of sex-sorted and non-sorted frozen-thawed stallion spermatozoa

In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen–thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197–201] was used to deposit low doses (6, 13 or 25 × 10⁶ frozen–thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500 IU, Intervet). Fertility was low, with one pregnancy (13 × 10⁶ spermatozoa, 500 μL) obtained after artificial insemination with frozen SS spermatozoa (n = 29 cycles) which resulted in the birth of a filly. Two pregnancies were obtained in mares inseminated with 6 × 10⁶ NS spermatozoa in 250 μL (n = 31 cycles). Mares failing to conceive on two experimental cycles were allocated to the conventional insemination group. Insemination with >500 × 10⁶ motile NS frozen–thawed spermatozoa, yielded satisfactory per cycle conception rates (35.5%, 22/62) for both stallions combined and was within the values of their normal fertility as quoted by the stud's records. This suggests that the quality of the frozen semen was acceptable and that the freezing processes yielded viable spermatozoa capable of fertilisation. The poor fertility after hysteroscopic insemination with low doses of sex-sorted or non-sorted spermatozoa from the same stallions may be directly attributable to the low dose insemination conditions with frozen–thawed rather than sex-sorted spermatozoa.