Effect of insemination with doses of 2 or 15 million frozen-thawed spermatozoa and semen deposition site on pregnancy rate in dairy cows

The effects of low-dose artificial insemination (AI) on pregnancy rates have seldom been studied in lactating dairy cows. We evaluated the pregnancy results after AI with doses of 2 and 15 million frozen-thawed spermatozoa and the effect of semen deposition in lactating dairy cows. A total of 284 first inseminations with 2 million spermatozoa and 312 first inseminations with 15 million spermatozoa were performed on 480 dairy farms. Low-dose inseminations (2 million spermatozoa) under field conditions in commercial dairy herds, without estrus synchronization, generally resulted in significantly reduced pregnancy rates compared with normal doses (15 million spermatozoa). The bull x technician effect on fertility was statistically significant. This finding indicates that there is a high variability in fertility among bulls using 2 million spermatozoa per dose. The semen deposition site did not influence pregnancy rates. It is concluded that a dose of 2 million frozen-thawed spermatozoa is probably too low for most bulls to achieve acceptable pregnancy rates in dairy cows.     

Longitudinal Study of Reproductive Performance of Female Cattle Produced by Somatic Cell Nuclear Transfer

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.     

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.     

Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo Spermatozoa

Advancements in reproductive technologies have shown seminal plasma (SP) as a nutritive-protective medium for spermatozoa metabolism, function and transport. At the same time quality variables and thus freezability of spermatozoa are influenced by SP proteins originating from male reproductive tract. One such protein, viz. PDC-109 is reported to influence freezability of spermatozoa in cattle. Thus the present investigation was designed to evaluate effect of seminal PDC-109 protein concentration on post-thaw cholesterol content and semen quality variables (SQP) as an indicator of membrane integrity and freezability, respectively of buffalo spermatozoa. Ejaculates (n = 42) selected on the basis of mass activity and individual motility were divided into three parts, first part for SP proteins isolation, second for cholesterol estimation and third part was cryo-preserved to evaluate freezability based on post-thaw SQP, viz. individual progressive motility, viability and acrosome integrity of spermatozoa. A total of 28 (66.7%) and 14 (33.3%) ejaculates from four bulls were found as freezable or non-freezable, respectively. Though total seminal plasma protein (TSPP) concentration was found similar in freezable and non-freezable ejaculates, the heparin binding proteins (HBP) content in non-freezable semen was greater (P < 0.01) than freezable ejaculates. There was a similar trend for the PDC-109 protein content in respective ejaculates. Cholesterol content of spermatozoa and SQP were greater (P < 0.05 and 0.01, respectively) in freezable as compared to non-freezable ejaculates of each bull at post-thaw stage. This study showed that concentrations of HBP and PDC-109 in non-freezable semen might be responsible for greater cryo-damage reflecting in poor freezability of buffalo spermatozoa.     

Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception

The present study was based on data obtained from artificial inseminations (AIs) performed with cryopreserved semen from elite bulls used in the Norwegian breeding program. Semen was diluted to standardize the number of spermatozoa to 18 million per AI dose. The aim of the study was to investigate whether the net sperm concentration at semen collection and the storage period in liquid nitrogen have any effect on probability of conception in dairy cattle. We demonstrated that the natural range of sperm concentration at semen collection within some of the bulls was associated with the probability of conception. However, no primary trend among bulls was found on the effect of sperm concentration at semen collection. This appears to be due to differences among bulls in their response to the dilution ratio of seminal plasma to extender. The effect of storage time was investigated in semen that had been stored between 1000 days and 2400 days in AI straws in liquid nitrogen at the AI center. Our findings showed that use of semen with the longest storage period, i.e. 1951-2400 days, resulted in a more than one percentage point lower probability of conception than semen with a shorter storage period. In conclusion, the net sperm concentration at semen collection, which affects the dilution ratio of seminal plasma to extender, should be considered individually among bulls to achieve optimal reproductive performance. Furthermore, this study gives support to the idea that a measurable degree of damage to the spermatozoa could occur during the preservation time in liquid nitrogen.     

Impaired semen quality of AI bulls fed with moldy hay: a case report

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay.     

Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers

This experiment was designed to test whether spermatozoa encapsulated in an alginate poly-L-lysine matrix had an extended fertile life in vivo after insemination. Estrus was synchronized in 417 primiparous Friesian and Jersey heifers with a system based on a CIDR-B intravaginal device before the heifers were inseminated either during proestrus (24 h after device removal) or at estrus (48 h after device removal). Pregnancy rates to first inseminations did not differ between the 24 and 48 h inseminations (61 vs 60.6%) with liquid semen diluted in Caprogen (control) but differed with encapsulated semen (45.1 vs 68.6%). The difference in pregnancy rates between the 2 types of semen was more pronounced (P < 0.08) in the animals that were visually detected in estrus. The mean survival time of spermatozoa in the female reproductive tract following insemination at the 24-h insemination time was estimated to be 50 +/- 7.5 h. The increased pregnancy rate with insemination of encapsulated spermatozoa at 48 h could have been due to this process predisposing spermatozoa to capacitate soon after insemination.     

Associations between insulin-like factor 3, scrotal circumference and semen characteristics in young Norwegian Red bulls

With the integration of genomic selection in the cattle artificial insemination (AI) industry, bulls are selected for their semen production capacity and fertility at a younger age than previously. Norwegian Red bull calves selected as candidates to become future AI bulls based on their genomic breeding value are kept in a performance testing station from around the age of 3–12 months, allowing for sample collection and analysis of different parameters during their pre- and peripubertal period. Insulin-like factor 3 (INSL3) is a small peptide hormone specifically secreted by the mature Leydig cells of the testes. In the foetus, it induces the first phase of testicular descent and is considered to reflect Leydig cell development during puberty; it could therefore be an interesting early indicator of future semen production capacity. The main objective of our study was to evaluate the relationship between INSL3, scrotal circumference (SC), and semen characteristics. This is the first time INSL3 was measured in the Norwegian Red population. We collected blood samples for analysis of INSL3 from 142 Norwegian Red bulls at the performance testing station and measured their SC on the same day. Altogether, measurements were made at four time points: upon arrival at the performance testing station (quarantine (Q): 2–5 months) and later at approximately 6, 9 and 12 months of age. Information on season and place of birth were made available from the database of the breeding company Geno, together with data on semen characteristics from the test station and the AI station. The median SCs for age groups Q, 6, 9, and 12 were 15, 21.5, 29, and 34 cm, respectively. INSL3 was shown to be positively correlated with SC (R = 0.4) but not with any of the semen characteristics. Similarly, we found no correlation between SC and sperm characteristics from data on ejaculates analysed at the performance testing station and AI station. The mean sperm volume for the 31 selected bulls with at least 10 ejaculates produced in the AI station increased from 2.3 ml at the performance testing station to 6.4 ml at the AI station. The corresponding increase in mean sperm concentration was from 497 million/ml to 1 049 million/ml. We conclude that INSL3 exhibits high inter-individual variability in the Norwegian Red bull population, which cannot be explained by the parameters measured in this study. At present, INSL3 cannot be used as a biomarker of sperm production in this breed.     

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas)

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean ± SD) 73.0 ± 12.2, 38.4 ± 8.8, 88.0 ± 0.1, and 59.3 ± 15.7%, respectively. A series of GnRH injections (3 x 250 μg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 ± 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 ± 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 ± 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 ± 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 ± 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.     

The dependence of the growth rate and meat content of young boars on semen parameters and conception rate

Boars have a decisive impact on the progress in pig production, however, there is no recent information about the optimal growth parameters during the rearing period for modern breed later used in artificial insemination (AI) stations. Therefore, the objective of the research was to conduct semen parameter and conception rate analyses on the basis of growth rate and meat content assessments made during the rearing of AI boars of different genotypes. The study was carried out between 2010 and 2014 and included 184 boars in five breed combinations: 46 Polish Large White, 50 Polish Landrace, 27 Pietrain, 36 Duroc×Pietrain and 25 Hampshire×Pietrain. Boars were qualified by daily gains and meat content assessment (between 170 and 210 days of life). A total number of 38 272 ejaculates were examined (semen volume (ml), spermatozoa concentration (×10⁶ ml⁻¹), total number of spermatozoa (×10⁹) and number of insemination doses from one ejaculate (n)). The fertility was determined by the conception rate (%). Semen volume, spermatozoa concentration and conception rate (P<0.01), followed by the total number of spermatozoa and insemination doses (P<0.05) were characterized by the highest variability in relation to breed of boars. The effect of daily gains was reported for spermatozoa concentration, number of insemination doses, conception rate (all P<0.01) and total number of spermatozoa (P<0.05). The peak of growth for spermatozoa concentration, total number of spermatozoa, insemination doses and conception rate was achieved for 800 to 850 g gains. Meat content affected semen volume, number of insemination doses and conception rate (P<0.05). Rearing boars while maintaining daily gains at the 800 to 850 g level and 62.5% to 65% meat content helps AI stations to increase the efficiency and economic profitability, and the number of insemination doses to increase by up to 300 doses/boar within a year. The analyses of growth parameters may help increase the efficiency and economic viability of AI stations.     

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.     

Estimation of genetic parameters of semen characteristics and reproductive traits in AI boars

(Co)variance components and further genetic parameters of boar semen characteristics and reproductive traits were estimated using the REML procedure applied to multi-trait animal models. The calculations were based on data from 210,733 ejaculates stemming from 2862 AI boars and collected from 1990 to 1997 in insemination stations for boars in the Czech Republic. Equal model equations for all traits included the AI station and the breed or breed combination as fixed effects, the interval between two collections for the boar as covariable and the animal and residual effects as random effects. The following heritabilities were estimated: semen volume 0.58, sperm concentration 0.49, progressive motion of spermatozoa 0.38, abnormal spermatozoa 0.34, number of total spermatozoa 0.42, number of insemination doses 0.40, number of piglets born alive 0.08, total number of piglets born 0.05 and conception rate 0.29. Heritabilities and genetic correlations were estimated on average values for each boar.     

A technique to artificially inseminate leafcutter ants

Artificial insemination (AI), the instrumental transfer of semen from the male to female reproductive organs, offers excellent opportunities to study mating system adaptations as it allows paternity to be experimentally manipulated. AI techniques have been developed for many animals, but rarely for ants, where they would be particularly useful as most species do not mate under controlled lab conditions. Here, we describe an AI technique for Atta leafcutter ants involving (1) the collection of ejaculates via induction of natural ejaculation, (2) storage in glass capillaries, and (3) transfer to queens using a modified AI equipment as used for honeybees. Queens were fixed and anesthetized in a queen holder, after which the sting chamber was opened with two steel hooks, the tip of the semen-containing glass capillary was inserted into the bursa copulatrix and the semen slowly expelled. Sperm was successfully stored in the spermatheca of queens, and some queens produced a small colony as a result. We could furthermore confirm the earlier observations that Atta semen is directly transferred to the spermatheca rather than to the bursa copulatrix as in most other eusocial insects. The technique that we present here can offer novel opportunities to study mating events such as sperm transfer, sperm competition, and cryptic female choice in ants. At present, the number of queens that produce colonies after AI remains low. However, this number will likely increase, as our results indicate that rearing conditions after AI influence colony founding success of artificially inseminated Atta queens.     

A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls

The artificial insemination (AI) industry is in need of an objective and rapid, but inexpensive method to evaluate frozen thawed bull semen ejaculates. This study presents a new fluorescence method that uses an automatized fluorometer and fluorophore stain propidium iodide that stains only those cells with damaged membranes. The fluorescence of the semen sample and the totally killed subsample were measured simultaneously, and viability was calculated. Every semen batch was analyzed before use in AI. For fertility evaluation, the nonreturn rates (NR%) obtained from 92,120 inseminations with the analyzed batches were recorded from 166 bulls (436 batches). This study confirms a 3.9% better NR% for the Finnish Holstein-Friesian breed than for Finnish Ayrshire. There was a clear seasonality in NR%: it differed (5.3%) significantly, being best in summer to autumn (June to October) and lowest in winter (January to March). The fluorometer method was fast and easy. The correlation between the total number of viable spermatozoa in an insemination dose and field fertility was low but significant (r = 0.051, P = 0.016), suggesting that the plasma membrane integrity evaluation can serve as a cost-beneficial quality control method of frozen-thawed semen at bull stations.     

Effect of a deep uterine insemination on spermatozoal accessibility to the ovum in cattle: a competitive insemination study

A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi-flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50x10(6) total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty-one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination.     

Assessment of the fertilizing potential of frozen bovine spermatozoa by in vitro induction of acrosome reactions with calcium ionophore (A23187)

The relationship between nonreturn rates of bulls in a commercial artificial insemination program and in vitro induction of acrosome reactions in frozen-thawed spermatozoa by the calcium ionophore, A23187, was investigated. Washed spermatozoa from 3 to 5 ejaculates, collected from each of 23 Holstein bulls, were incubated for 1 h with 1 microM A23187. Acrosome reactions were determined by fluorescence microscopy. The percentage of increase in acrosome reaction in the ionophore-treated compared with control samples was significantly correlated to the 90-d nonreturn rate of the bulls (r = 0.86; P < 0.001). In a second experiment, a significant correlation was obtained between the fertility of bulls predicted on the basis of induced acrosome reaction and achieved 90-d nonreturn rate (r = 0.84; P < 0.005). No other assessments of semen quality (post-freezing motility, percentage of morphologically normal spermatozoa) was significantly correlated with fertility. Finally, the regression between acrosome reaction induction obtained from young bulls was used to predict the fertility of mature bulls whose semen was in widespread use (actual versus predicted nonreturn rate, r = 0.88; P < 0.0001).     

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.     

Effect of season on fertility of frozen buffalo semen

Twenty double ejaculates from each of ten water-buffalo bulls were collected in June (non-breeding season) and again in November (breeding season). Fresh semen was screened for sperm quantity, motility, eosin uptake, and sperm morphology and was frozen using lactose, skim-milk, and Tris extenders. Thawed semen was checked for motility and Sephadex filtration. Half of each semen batch was used for artificial insemination in the breeding season and the other half during the non-breeding season.Laboratory screening revealed that June semen had a significantly lower Sephadex filtration rate and a higher percentage of abnormal sperm cells, and three June ejaculates were excluded from further processing due to poor sperm motility. In the remaining ejaculates the motility before freezing and the sperm cell quantity were higher in June semen than in November semen. Eosin uptake, mass motility, and post-freeze-motility did not vary with season. November semen produced significantly higher pregnancy rates than June semen over a total of 3220 inseminations in both seasons. Forty percent of the observed seasonality of buffalo fertility was attributable to the male. No fertility differences appeared between extenders used. When November semen was used, the fertility in adult buffaloes in both seasons was higher than in heifers.     

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis)

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.