Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.     

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = −0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.     

Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls

We investigated fluorometry to study sperm viability and flow cytometry to study sperm chromatin structure. We also assessed sperm quality after thawing relative to field fertility after AI as shown by 56-day non-return rates (56-d NRR) Frozen-thawed semen samples were obtained from 20 Swedish Red and White bulls (1 to 3 semen batches/bull) and the fertility data were based on 6,369 AIs. Fluorometry enabled simultaneous detection of sperm viability and concentration in Hoechst 33258-stained semen samples. Sperm chromatin structure assay (SCSA) evaluated denaturability of sperm nuclear DNA in situ after acid treatment. The intensity of fluorescence in non-permeabilized samples was negatively (r = -0.60, P < 0.001) correlated with microscopically-assessed sperm viability, and the fluorescence of permeabilized semen samples significantly (r = 0.67, P < 0.001) correlated with sperm concentration as assessed by hemocytometry. From the fluorescence output, the calculated percentage of damaged cells was negatively (r = -0.71, P < 0.001) correlated with the number of live cells derived from the microscopic assessment of sperm viability and concentration. This variable was significantly correlated with fertility results both at batch (r = -0.39, P < 0.05), and bull (r = -0.57, P < 0.01) levels. The SCSA variables SDalphat and COMPalphat were significantly (r = -0.59-0.64, P < 0.001) correlated with sperm viability variables after thawing but only the COMPalphat correlated significantly (r = -0.53, P < 0.05) with fertility results and solely at the bull level. The results indicate that fluorometric assessment is in good agreement with other practiced procedures and can be performed with sufficient accuracy. The SCSA may be a valuable complement for routinely practiced microscopic evaluation of sperm morphology of AI bull semen     

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.     

Functional integrity of sex-sorted, frozen-thawed boar sperm and its potential for artificial insemination

Despite being developed in its present form 20 years ago, sex-sorting of mammalian sperm is still a work in progress. While relatively successful in cattle and sheep, the unique challenges of incorporating sex-sorted sperm into pig production have not yet been overcome. Generally speaking, boar sperm survive freeze-thawing less well and are required in larger numbers for insemination, while in vitro embryo production of pig embryos is less successful compared to other domestic species [Niemann H, Rath D. Progress in reproductive biotechnology in swine. Theriogenology 2001;56:1291-1304]. Due to the large number of sperm required for artificial insemination in pigs, a technique of storing sperm after sorting must be developed while adequate numbers of sperm are allocated into X- or Y-chromosome-bearing enriched pools. Cryopreservation is perhaps the ideal method of storage between sorting and insemination, as it allows unlimited time to build up a sperm bank, whereas liquid-storage requires the use of sperm within days of sorting. The limited number of studies investigating the survivability of sex-sorted, frozen-thawed boar sperm have produced promising in vitro results but poor in vivo outcomes. Before fertility can be improved, the causes of any damage to sperm function during the sex-sorting and freeze-thawing procedures must be more fully understood. Once defined, the source of damage may be minimised and this would lead to increased success rates after in vivo application of sex-sorted, frozen-thawed boar sperm.     

Field fertility of frozen-thawed boar sperm at low doses using non-surgical, deep uterine insemination

The lowest dose of frozen-thawed boar sperm used for deep uterine artificial insemination (DUI) of sows has been 100x10(6). A three stage field study was performed to establish to what level the dose of frozen-thawed sperm used for DUI could be reduced without adversely affecting the fertility of the sow. In stage 1, 15 sows were inseminated twice with 1000x10(6) fresh or frozen-thawed sperm at 24 and 36 h post-detection of oestrus. In stage 2, 262 sows were inseminated with 62.5, 250 or 1000x10(6) fresh or frozen-thawed sperm at 24, 36, or 24 and 36 h after detection of oestrus. Stage 3 involved post mortem investigation of the uterine lining to assess damage caused by insertion of the insemination catheter. All sows inseminated in stage 1 of the study farrowed. In stage 2, the non-return (NRR) and farrowing rates of each group were compared to a control double cervical insemination of 3250x10(6) fresh sperm. As few as 62.5x10(6) fresh sperm could be deposited at a single insemination without reduction in NRR or farrowing rates compared with the control group. A double DUI with 250x10(6) frozen-thawed sperm was required before fertility was equivalent to the controls. Investigation of the uterine lining after insertion of the DUI catheter revealed evidence of bleeding, warranting further investigation of the viability of widespread use of the Firflex catheter, despite the promising fertility achieved here with low doses of spermatozoa.     

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.     

Timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm

The objective was to evaluate the effects of timing of insemination and type of semen in cattle subjected to timed artificial insemination (TAI). In Experiment 1, 420 cyclic Jersey heifers were bred at either 54 or 60 h after P4-device removal, using either sex-sorted (2.1 × 10⁶ sperm/straw) or non-sorted sperm (20 × 10⁶ sperm/straw) from three sires (2 × 2 factorial design). There was an interaction (P = 0.06) between time of AI and type of semen on pregnancy per AI (P/AI, at 30 to 42 d after TAI); it was greater when sex-sorted sperm (P < 0.01) was used at 60 h (31.4%; 32/102) than at 54 h (16.2%; 17/105). In contrast, altering the timing of AI did not affect conception results with non-sorted sperm (54 h = 50.5%; 51/101 versus 60 h = 51.8%; 58/112; P = 0.95). There was an effect of sire (P < 0.01) on P/AI, but no interaction between sire and time of AI (P = 0.88). In Experiment 2, 389 suckled Bos indicus beef cows were enrolled in the same treatment groups used in Experiment 1. Sex-sorted sperm resulted in lower P/AI (41.8%; 82/196; P = 0.05) than non-sorted sperm (51.8%; 100/193). In addition, there was a tendency for greater P/AI (P = 0.11) when TAI was performed 60 h (50.8%; 99/195) versus 54 h (42.8%; 83/194) after removing the progestin implant. In Experiment 3, 339 suckled B. indicus cows were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ultrasonographic examinations were performed twice daily in all cows to confirm ovulation. On average, ovulation occured 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. The P/AI was greatest (37.9%) for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly less for TAI performed between 12.1 and 24 h (19.4%) or >24 h (5.8%) before ovulation. In conclusion, sex-sorted sperm resulted in a lesser P/AI than non-sorted sperm following TAI. However, improvements in P/AI with delayed time of AI were possible (Experiments 1 and 3), and seemed achievable when breeding at 60 h following progestin implant removal, compared to the standard 54 h normally used in TAI protocols.     

Enriching the captive elephant population genetic pool through artificial insemination with frozen-thawed semen collected in the wild

The first successful AI in an elephant was reported in 1998, using fresh semen. Since then almost 40 calves have been produced through AI in both Asian and African elephants worldwide. Following these successes, with the objective of enriching the captive population with genetic material from the wild, we evaluated the possibility of using frozen-thawed semen collected from wild bulls for AI in captivity. Semen, collected from a 36-yr-old wild African savanna elephant (Loxodonta africana) in South Africa was frozen using the directional freezing technique. This frozen-thawed semen was used for four inseminations over two consecutive days, two before and two after ovulation, in a 26-yr-old female African savanna elephant in Austria. Insemination dose of 1200 × 10⁶ cells per AI with 61% motility resulted in pregnancy, which was confirmed through ultrasound examination 75, 110 and 141 days after the AI procedure. This represents the first successful AI using wild bull frozen-thawed semen in elephants. 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, as was done in this study, between wild and captive populations, without the need to transport stressed or potentially disease-carrying animals or to remove animals from the wild. 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 genetic diversity management and phenotype selection in these endangered mammals.     

Influence of season on characteristics of the ejaculate from bulls in an artificial insemination centre in Nigeria

The influence of season on the ejaculate characteristics of Zebu, Friesian and their crossbred bulls in an A.I. programme in Nigeria was investigated over a 2-year period.Ejaculate volume, sperm concentration, percent morphologically normal spermatozoa and percent live spermatozoa were significantly higher in the rainy season than in the dry season. Total spermatozoa per ejaculate averaged 3.32 × 10⁹ and 10.10 × 10⁹ for the dry and rainy seasons respectively. Corresponding proportions of total morphologically defective spermatozoa per ejaculate were 14.05% and 6.46%. Percent live spermatozoa were 82.34% and 84.61% while the corresponding sperm concentrations were 0.97 × 10⁹ rmand 1.74 × 10⁹ for the dry and rainy seasons respectively. All differences were statistically significant (P < 0.05).Ejaculate quality was better during the rainy season. Consequently semen collected and frozen during the rainy season may produce higher fertility rates in an A.I. programme.     

Early pregnancy loss in sows after low dose, deep uterine artificial insemination with sex-sorted, frozen-thawed sperm

Recent developments in reproductive technologies have enabled the production of piglets of a predetermined sex via non-surgical, low dose artificial insemination. The practical application of sex-sorting technology to the pig is made challenging by the large numbers of sperm required for successful insemination of sows. One way of overcoming the time required for sex-sorting may be to create a bank of cryopreserved, sex-sorted sperm, thus making available appropriate doses as sows require insemination. To date, little success has been achieved with non-surgical inseminations of sex-sorted boar sperm. This study attempted to achieve litters of a predetermined sex after a double insemination of sows with 160x10(6) sex-sorted, frozen-thawed sperm. Sows were synchronised and sperm were non-surgically inseminated into the proximal third of the uterine horn at 36 and 42 h after hCG administration. Sows inseminated with sex-sorted sperm achieved similar pregnancy rates to those receiving an equal dose of unsorted, frozen-thawed sperm. However, all sows conceiving after insemination with sex-sorted sperm returned to oestrus within 57 days of insemination. This was a higher rate of pregnancy loss than observed for sows inseminated with unsorted sperm (37.5%; P=0.031). A combination of low sperm numbers and potentially compromised developmental capability of embryos derived from sex-sorted sperm may have resulted in this early stage loss of pregnancy.     

Relationships among fertility, scrotal circumference, seminal quality, and libido in Santa Gertrudis bulls

Forty Santa Gertrudis bulls were used to examine relationships among scrotal circumference, seminal quality, libido, and fertility [assessed as the percent pregnant of estrous females (PE rate) and the percent pregnant of females mated (PM rate)]. These bulls were selected from 220 two year old bulls to represent variations in scrotal circumference and seminal quality. Each of the 40 bulls were exposed to 100 cyclic Santa Gertrudis heifers for a 4-day (96 hr) breeding period. The number of estrous females available to each bull varied from 12 to 27. A breeding soundness examination (BSE) was conducted on each bull approximately 45 days prior to the 4-day breeding period and immediately after the 4-day breeding period. The three components of the BSE scroe (scrotal circumference, spermatozoal abnormalities and spermatozoal motility) were not significantly correlated with PE rate or PM rate at either evaluation. There was no significant correlation between PM rate and scrotal circumference; however, each of the 4 bulls having a scrotal circumference less than 30 cm had a PM rate below 31%. Relationships between seminal quality and PM rate were unclear and differed between the two evaluations. There was a trend for bulls having poor seminal quality at the first evaluation to improve by the second evaluation. Consequently, fluctuations in seminal quality between evaluations is one possible explanation for low correlations between seminal parameters and PM rate. Libido (number mated/number in estrus x 100) was positively correlated (r = 0.44) with PE rate. Using a stepwise regression procedure, the independent variables accounting for the most variation in PE rate (dependent variable) included libido, secondary spermatozoal abnormalities, and BSE score (r(2) = 0.44). Results of this study indicate that current methods of fertility evaluation did not accurately predict the fertility of individual Santa Gertrudis bulls as measured by PE rate and PM rate during a 4 day breeding period.     

Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in Sweden

We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 degrees C, at 4.2 degrees C/min (control split sample) or at 0.1 degree C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 m.o. or 66 to 79 m.o. were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinin [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6% units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa--average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL)--assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested.     

Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes

Fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) were used to simultaneously evaluate the integrity of plasma and acrosomal membranes as well as mitochondrial membrane function in cryopreserved bovine semen and to verify its influence on fertility and postinsemination uterine vascularization. One hundred eighty-two Nellore cows were distributed for artificial insemination (AI) using semen batches separated according to the cell percentage presenting intact plasma membrane, intact acrosome, and high mitochondrial function (IPIAH): group G (44.5% IPIAH, n = 68), group M (23.0% IPIAH, n = 56), and group R (8.5% IPIAH, n = 58). The uterine hemodynamic was evaluated by Doppler sonogram in three periods: 30 hours before AI, 4 and 24 hours after AI were considered the resistance index and the uterine vascularization score. The pregnancy rate of group G (64.7%) was greater (P > 0.05) compared with group R (36.2%), but both did not differ from group M (50.0%). There was no effect (P > 0.05) of semen quality on uterine vascularization. Greater vascularization was noticed 4 hours after AI than 30 hours before and 24 hours after AI. Semen evaluation using fluorescent probes contributes to predicting fertilizing potential of semen. The use of semen with less percentage of IPIAH sperm does not alter uterine hemodynamic in cows.     

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.     

Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls

The performance of frozen-thawed spermatozoa from 10 Holstein bulls in a range of in vitro diagnostic tests and the relationship with adjusted in vivo fertility data was determined. The tests included an assessment of motility (subjective and computer-assisted), morphology, concentration, viability, acrosomal and chromatin integrity conducted immediately post-thaw and after swim-up, in conjunction with membrane status (CTC staining) and migration in an artificial cervical mucus. Adjusted in vivo fertility correlated with subjectively assessed post-thaw motility (r=0.672, p=0.033), post-thaw straight-line velocity (r=0.636, p=0.048), post-thaw sperm morphology (r=-0.762, p=0.010), post-thaw sperm viability (r=0.635, p=0.048), the concentration of spermatozoa after swim-up (r=0.649, p=0.042), sperm morphology after swim-up (r=-0.687, p=0.028), the number of spermatozoa migrating 10mm into artificial cervical mucus (r=0.632, p=0.050) and the distance migrated by the vanguard spermatozoon in artificial mucus (r=0.701, p=0.024). A stepwise regression analysis identified tests which, when combined, produced models with a strong correlation (R(2)>0.9) to fertility.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

Development of an artificial insemination protocol in llamas using cooled semen

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose-egg yolk (L-EY), (2) Tris-citrate-fructose-egg yolk (T-F-EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5 h and remained at that temperature during 24 h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L-EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A-D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa, cooled to 5°C and kept for 24 h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24 h. These groups (A-C) were inseminated between 22 and 24 h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24 h and AI was carried out within 2 h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation.     

Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls

Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves.     

Relationships among spermatozoal abnormalities and the selenium concentration of blood plasma,semen, and reproductive tissues in young bulls

The concentration of selenium (Se) was measured by instrumental neutron activation analysis in samples of blood plasma, semen, and reproductive and non-reproductive tissues obtained from each of 12 young bulls (8 Angus and 4 Simmental). Semen was collected by electro-ejaculation and used for measurements of the frequency of primary and secondary spermatozoal abnormalities. The mean Se concentration (μg/ml) of semen (± SD) was 0.461 (±0.223) compared to 0.061 (±0.014) for blood plasma. Tissue from the testis, caput epididymis and cauda epididymis had mean Se concentrations of from 2.5 to 2.7 μg/g compared to less than 1.8 μg/g in all other tissues except the pituitary gland (3.4 μg/g) and kidney cortex (6.9 μg/g).Correlations of the proportional incidence of spermatozoal abnormalities with Se concentrations in reproductive tissues, semen or blood plasma were low. Although Se may be concentrated in the testis and epididymis, the Se concentration was not related to spermatozoal abnormalities.