First successful artificial insemination with frozen-thawed semen in rhinoceros

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of ∼135 × 10⁶ motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of ∼500 × 10⁶ motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals.     

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm,Bombyx mori

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.     

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10⁶ sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.     

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves,straw sizes,and thawing rates

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (−10 °C/min) or a fast (−40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8 s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P < 0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P > 0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P < 0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P > 0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples thawed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P < 0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.     

Timed artificial insemination should be performed early when used norgestomet ear implants are applied for synchronizing ovulation in beef heifers

The present study evaluated the effect of the type of norgestomet ear implant (new vs. used) on the ovarian follicular response (experiment 1) and pregnancy per artificial insemination (AI) (P/AI; experiment 2) of beef heifers subjected to an estradiol plus progestin timed artificial insemination (TAI) program. In experiment 1, 57 cyclic beef heifers were randomly assigned to one of two groups according to the type (new or previously used for 9 days) of norgestomet ear (NORG) implant. At the time of NORG implant insertion, the heifers were treated with 2 mg of intramuscular estradiol benzoate. Eight days later, the NORG implants were removed, and the heifers received an intramuscular administration of 150 μg of d-cloprostenol, 300 IU of equine chorionic gonadotropin, and 0.5 mg of estradiol cypionate. The heifers had their ovaries scanned every 12 hours from the time of NORG implant removal to 96 hours after verifying the occurrence and timing of ovulation. No difference (P = 0.89) was observed in the ovulation rates between the two treatments (new = 80.0%; 24/30 vs. used = 81.5%; 22/27). However, the heifers treated with a used NORG implant had (P = 0.04) higher proportion (36.4%; 8/22) of early ovulation (between 36 and 48 hours after NORG implant removal) compared with the heifers treated with a new NORG implant (8.3%; 2/24). In experiment 2, at the beginning of the synchronization protocol, 416 beef heifers were randomly assigned into two groups, as described in the experiment 1. Two days after the NORG implant removal, the heifers were reassigned to be inseminated at 48 or 54 hours after NORG implant removal. There was an interaction (P = 0.03) between the type of NORG implant and the timing of TAI on P/AI. The timing of insemination only had an effect (P = 0.02) on the P/AI when the heifers were treated with a used NORG implant [(TAI 54 hours = 41.9% (44/105) vs. TAI 48 hours = 58.6% (58/99)]. In conclusion, beef heifers synchronized with a used NORG implant plus estradiol exhibited a higher proportion of earlier ovulations, and TAI in these heifers should be performed 48 hours after removal of used NORG implants.     

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10⁶ spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.     

Resynchronization with unknown pregnancy status using progestin-based timed artificial insemination protocol in beef cattle

Two experiments were designed to evaluate the use of resynchronization (RESYNCH) protocols using a progestin-based timed artificial insemination (TAI) protocol in beef cattle. In experiment 1, 475 cyclic Nelore heifers were resynchronized 22 days after the first TAI using two different inducers of new follicular wave emergence (estradiol benzoate [EB; n = 241] or GnRH [n = 234]) with the insertion of a norgestomet ear implant. At ear implant removal (7 days later), a pregnancy test was performed, and nonpregnant heifers received a dose of prostaglandin plus 0.5 mg of estradiol cypionate, with a timed insemination 48 hours later. The pregnancy rate after the first TAI was similar (P = 0.97) between treatments (EB [41.9%] vs. GnRH [41.5%]). However, EB-treated heifers (49.3%) had a greater (P = 0.04) pregnancy per AI (P/AI) after the resynchronization than the GnRH-treated heifers (37.2%). In experiment 2, the pregnancy loss in 664 zebu females (344 nonlactating cows and 320 cyclic heifers) between 30 and 60 days after resynchronization was evaluated. Females were randomly assigned to one of two groups (RESYNCH 22 days after the first TAI [n = 317] or submitted only to natural mating [NM; n = 347]). Females from the NM group were maintained with bulls from 15 to 30 days after the first TAI. The RESYNC-treated females were resynchronized 22 days after the first TAI using 1 mg of EB on the first day of the resynchronization, similar to experiment 1. No difference was found in P/AI (NM [57.1%] vs. RESYNC [61.5%]; P = 0.32) or pregnancy loss (NM [2.0%] vs. RESYNC [4.1%]; P = 0.21) after the first TAI. Moreover, the overall P/AI after the RESYNCH protocol was 47.5%. Thus, the administration of 1 mg of EB on day 22 after the first TAI, when the pregnancy status was undetermined, promotes a higher P/AI in the resynchronized TAI than the use of GnRH. Also, the administration of 1 mg of EB 22 days after the TAI did not affect the preestablished pregnancy.     

Controlling contagious agalactia in artificial insemination centers for goats and detection of Mycoplasma mycoides subspecies capri in semen

Many goat artificial insemination (AI) centers in Spain have adopted new measures to control contagious agalactia (CA). To avoid the introduction of male goats carrying mycoplasma organisms subclinically in their external ear canal (auricular carriers) in these centers, two ear swabs and a blood sample are obtained from all candidate animals for polymerase chain reaction (PCR), culture (swabs) and serologic tests to detect the presence of mycoplasmas. In addition, the semen produced at these centers is routinely cultured and PCR tested also to detect the presence of mycoplasmas. One y after the introduction of this program, we tested 48 ear swabs and 24 blood samples from 24 candidates for admission to these AI Centers. Three of these ear swab samples (3/48, 6.25%) scored positive for the presence of mycoplasmas; Mycoplasma agalactiae (Ma) was detected in two samples and Mycoplasma mycoides subsp. capri (Mmc) in one. All animals were serologically negative for Ma. Also, out of 173 semen samples obtained from 137 admitted animals (2 and 3 samples were obtained in 16 and 10 bucks, respectively), one (1/173, 0.56%) was positive for Mmc. Our findings suggest that ear swab and semen samples are useful tools to control CA at AI Centers. The introduction of this program has also resulted in the first detection of Mmc in semen from a naturally infected goat, confirming the ability of this mycoplasma to colonize the reproductive tract of male goats. These results highlight the need to improve control measures in semen producing centers to minimize the risk of CA transmission.     

A genetic linkage map for the apicomplexan protozoan parasite Eimeria maxima and comparison with Eimeria tenella

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of ∼14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10 years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines.     

Ultrasonographic and laparoscopic evaluation of the reproductive tract of the captive female African lion (Panthera leo)

The use of transabdominal ultrasonography to assess the oestrous cycle has not been previously described in the African lion (Panthera leo). Twelve sexually mature lionesses and five female cubs had their reproductive organs assessed by transabdominal ultrasound. Ovarian findings were compared to laparoscopic findings while performing laparoscopic ovariectomy or salpingectomy. Vaginal cytology was performed and serum progesterone levels were determined. By combining all data the oestrous cycle stage of each lion was determined. One lion was far pregnant and was not operated on. In adults a uterine body could be seen ultrasonographically in 67% of lions while mural structures could be distinguished in 44% of lions. Five uterine horns could be seen in 3 lions. In 12 adults 10 ovaries were found of which eight had discernable follicles or luteal structures. During laparoscopy 12 active ovaries were seen with luteal structures seen in 11 ovaries and follicles in 2 ovaries. Using laparoscopy as the gold standard, ultrasonography had a sensitivity of 66% and specificity of 83% to detect ovarian reproductive activity. Two uterine cysts and a cluster of periovarian cysts were seen in three different lions. Three lions were pregnant, two were in oestrus, three in a luteal phase (dioestrus), and four were in anoestrus. Transabdominal ultrasound in combination with serum progesterone levels and vaginal cytology can be used to assess ovarian cyclical activity with reasonable accuracy in captive bred lions.     

Improvement of the cryopreservation of the fungal starter Geotrichum candidum by artificial nucleation and temperature downshift control

Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.     

Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows II: Strategies and factors affecting fertility

In Experiments 1, 2, and 3, we evaluated the effects of temporary weaning (TW), equine chorionic gonadotropin (eCG), and follicle-stimulating hormone (FSH) treatments on results of a fixed-time artificial insemination (TAI) protocol in postpartum Bos indicus cows. In Experiment 1, treatment with 400 IU eCG or with TW for 48 h consistently improved pregnancy rates (PRs) at TAI, but, in Experiment 2, FSH treatment was less effective than eCG or TW. In Experiment 3, the inclusion of eCG treatment in cows subjected to TW did not improve PRs. We concluded that TW or 400 IU eCG should be included in the TAI protocol in postpartum Bos indicus cows to enhance fertility. In Experiment 4, we used records from heifers and cows treated with the proposed protocol during the 2006-2007 (n = 27,195) and 2007-2008 (n = 36,838) breeding seasons from multiple locations in Brazil to evaluate factors potentially affecting PRs. Overall PR at TAI was 49.6% (31,786 of 64,033). Pregnancy rate differed (P < 0.01) among farm within location (results ranging between 26.8% and 68.0%; P < 0.01), cow group within farm, by breed (Bos indicus, 48.3% [26,123 of 54,145]; Bos taurus, 61.7% [3652 of 5922]; and crossbred Bos indicus × Bos taurus, 50.7% [2011 of 3966]), category (nulliparous, 39.6% [2095 of 5290]; suckled primiparous, 45.2% [3924 of 8677]; suckled multiparous, 51.8% [24,245 of 46,767]; and nonsuckled multiparous, 46.1% [1522 of 3299]), body condition score at TAI (≤2.5, 43.0% [3409 of 7923]; 3.0, 49.6% [18,958 of 38,229]; and ≥3.5, 52.7% [9419 of 17,881]). Days postpartum at beginning of protocol did not affect PR (30 to 60 d, 47.6% [4228 of 8881]; 61 to 90 d, 51.7% [16,325 to 31,572]; and 91 to 150 d, 50.8% [7616 to 14,991]; P > 0.1). Pregnancy rate was also consistently affected (P < 0.01) by sire (results ranging from 7.2% to 77.3%) and artificial insemination technician (results ranging from 15.1% to 81.8%).     

Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain

The genetic diversity and prevalence of virtually all Theileria and Babesia species in a sheep population were studied using a specifically designed reverse line blot macroarray. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against generic and species-specific probes. In a first screening (Study I), 320 apparently healthy animals corresponding to 32 flocks located in the Basque Country (Northern Spain) were analysed. The survey demonstrated a high prevalence of subclinical infections (64.7%). Three Theileria genotypes were identified, sharing 96.7-97.0% similarity between their 18S rRNA gene sequences: Theileria ovis, Theileria sp. OT1 (99.6% similarity with the recently described pathogenic piroplasm Theileria sp. China 1), and Theileria sp. OT3. Two Babesia species sharing 91.5% similarity were also detected: Babesia ovis and Babesia motasi. The complete 18S rRNA gene sequences of these and other piroplasm species were phylogenetically analysed. Prevalence of piroplasms was also investigated in a second group of 80 sheep from 16 flocks reared in mountain areas that had been heavily exposed to ticks and had suffered a recent abortion episode (Study II). The screening revealed a significantly higher (P < 0.05) prevalence (78.7%) of piroplasm infections compared to Study I. Although the prevalence rates for some piroplasm species were significantly related to abortion (e.g. Theileria sp. OT3), decreases in the red cell parameters were not significant. The widespread distribution of Theileria spp. in the studied sheep population suggests that the parasites involved are of relatively low pathogenicity, in contrast to what has been reported for Theileria sp. China 1 in other countries.