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

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

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.     

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus)

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n = 81) were inseminated into the uterus by rectum manipulation with 1 × 10⁶ (X1 and Y1 group, respectively) or 2 × 10⁶ (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 × 10⁷ non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700 IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P > 0.05), but higher for control (75%) treatment (P < 0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P < 0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250 d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.     

Birth of domestic cat kittens of predetermined sex after transfer of embryos produced by in vitro fertilization of oocytes with flow-sorted sperm

Our goals were to: (1) determine if domestic cat sperm could be sorted to high purity by flow cytometry after overnight shipment of cooled samples; (2) evaluate the efficiency with which sorted sperm could be used to generate cat embryos in vitro; and (3) determine if live kittens of predetermined sex could be produced after transfer of embryos derived by IVF using sorted sperm. Semen samples (n = 5) from one male were extended in electrolyte-free solution and shipped overnight at 4 °C to the sorting facility. Samples were adjusted to 75 × 10⁶ sperm/mL and stained with Hoechst 33342. After 1 h at 34.5 °C, samples were adjusted to 50 × 10⁶ sperm/mL with 4% egg yolk TALP + 0.002% food dye and sorted by high-speed flow cytometry. Later resort analysis confirmed purities of 94% and 83% for X- and Y-chromosome bearing sperm, respectively. Sorted sperm were centrifuged, re-suspended in TEST yolk buffer and shipped overnight to the IVF laboratory. After IVF of in vivo matured oocytes with X-chromosome bearing sperm, cleavage frequency was 62% (54/87). After IVF of IVM oocytes with control, X- or Y-chromosome bearing sperm, the incidence of cleavage was 42% (48/115), 33% (40/120), and 35% (52/150), respectively, and blastocyst development was 53% (21/40), 50% (11/22), and 55% (23/42), respectively (P > 0.05). On Day 2, 45 embryos produced by IVF of in vivo matured oocytes with X-chromosome bearing sperm were transferred to the oviduct of four Day 1 recipients, three of which subsequently delivered litters of one, four, and seven female kittens, respectively. In conclusion, we confirmed that sperm sorting technology can be applied to domestic cats and established that kittens of predetermined sex can be produced.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Comparison of efficiency between two artificial insemination methods using frozen–thawed semen in domestic cat (Felis catus): Artificial insemination in domestic cats

The aim of this study was to compare the efficiency of the intravaginal (IVAI) vs. intrauterine artificial insemination (IUAI) using frozen–thawed sperm in the domestic cat. Semen was collected from two tom cats using an artificial vagina and samples were assessed for motility (computer-assisted sperm analysis (CASA)), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP/YOLK (4% of glycerol), sperm samples were loaded into 0.25 mL straws (25 × 10⁶ motile sperm/straw), incubated at 5 °C for 20 min and cryopreserved over liquid nitrogen (LN₂) vapor for 15 min and then immersed in LN₂. For each AI, four straws from the same male were thawed (12 s at 46 °C) and centrifuged at 250 × g for 8 min to pellet the sperm. The supernatant was discarded and sperm pellet resuspended with the remaining liquid, approximately 100 μL, and analyzed as described above. Queens were treated with a single im injection of 100 IU eCG to induce ovarian follicular development. Final oocyte maturation and ovulation was induced with 100 IU hCG given im at 82–84 h after eCG administration. Thirty hours after hCG administration, females were inseminated either intrauterine (n = 8 queens) or intravaginally (n = 8 queens), using thawed sperm from a single male. Although a pronounced decrease in sperm motility, acrosome and plasma membrane integrity was observed in sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method compared with 0% pregnancy when inseminated intravaginally.     

In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations

The acrosome reaction (AR) is a prerequisite for normal sperm fertilizing capability and can be studied in vitro after induction by various agents. The efficacy of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential. During the past two decades, a number of attempts have been made to relate the in vitro-induced AR to field fertility in several species. However, to our knowledge, no studies have combined in vitro induction of the AR with the simultaneous detection of sperm viability and acrosomal status using a high-precision flow cytometric technique. Furthermore, large-scale fertility trials using low-dose inseminations are pending. In the current study, the relationship between field fertility and the in vitro-induced AR was investigated using three ejaculates from each of 195 bulls, 156 Holstein and 39 Jersey bulls (Bos taurus), participating in a progeny test program including low-dose inseminations. A range of insemination doses, varying from 2.0 × 10⁶ to 15 × 10⁶ sperm/dose, was obtained by a controlled dilution process applied to each ejaculate. Different insemination doses were distributed at random among 75,610 experimental first inseminations in 4721 herds and 208 artificial insemination (AI) technicians. Simultaneous detection of sperm viability and acrosomal status was achieved using a triple color flow cytometric technique. Sperm samples from the bulls displayed a wide range of ability to acrosome react in response to calcium ionophore A23187. Both reproducibility of the AR response after induction and relationship between ability to acrosome react and field fertility was highly dependent on the definition of AR inducibility. Six basic and six combined AR indices were assessed. The AR index expressing the fraction of acrosome reacted sperm in the live sperm population after induction by ionophore had the highest repeatability, best described the biological variation in the studied population, and yielded the best significant predictive values on field fertility among the 12 indices considered. Moreover, the ability of sperm to acrosome react appeared to be a noncompensable trait that affects fertility regardless of the number of sperm per insemination dose. The current results therefore indicate that this sperm parameter is important in the field and also may play a role in the IVF laboratory.     

The rate of oxygen utilization by cells

The discovery of oxygen is considered by some to be the most important scientific discovery of all time—from both physical-chemical/astrophysics and biology/evolution viewpoints. One of the major developments during evolution is the ability to capture dioxygen in the environment and deliver it to each cell in the multicellular, complex mammalian body—on demand, i.e., just in time. Humans use oxygen to extract approximately 2550 calories (10.4 MJ) from food to meet daily energy requirements. This combustion requires about 22 mol of dioxygen per day, or 2.5 × 10^− 4 mol s^− 1. This is an average rate of oxygen utilization of 2.5 × 10^− 18 mol cell^− 1 s^− 1, i.e., 2.5 amol cell^− 1 s^− 1. Cells have a wide range of oxygen utilization, depending on cell type, function, and biological status. Measured rates of oxygen utilization by mammalian cells in culture range from < 1 to > 350 amol cell^− 1 s^− 1. There is a loose positive linear correlation of the rate of oxygen consumption by mammalian cells in culture with cell volume and cell protein. The use of oxygen by cells and tissues is an essential aspect of the basic redox biology of cells and tissues. This type of quantitative information is fundamental to investigations in quantitative redox biology, especially redox systems biology.     

Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos

The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human α1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P < 0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V⁻/PI⁻), early apoptotic (Ann-V⁺/PI⁻), and late apoptotic/early necrotic sperm (Ann-V⁺/PI⁺) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V⁻/PI⁺) was significantly different (P < 0.05) between transgenic and non-transgenic boars (3.4% ± 2.7; 7.2% ± 2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r = 0.9787; P < 0.0001).     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Mice lacking FABP9/PERF15 develop sperm head abnormalities but are fertile

The male germ cell-specific fatty acid-binding protein 9 (FABP9/PERF15) is the major component of the murine sperm perforatorium and perinuclear theca. Based on its cytoskeletal association and sequence homology to myelin P2 (FABP8), it has been suggested that FABP9 tethers sperm membranes to the underlying cytoskeleton. Furthermore, its upregulation in apoptotic testicular germ cells and its increased phosphorylation status during capacitation suggested multiple important functions for FABP9. Therefore, we investigated specific functions for FABP9 by means of targeted gene disruption in mice. FABP9^−/− mice were viable and fertile. Phenotypic analysis showed that FABP9^−/− mice had significant increases in sperm head abnormalities (~ 8% greater than their WT cohorts); in particular, we observed the reduction or absence of the characteristic structural element known as the “ventral spur” in ~ 10% of FABP9^−/− sperm. However, deficiency of FABP9 affected neither membrane tethering to the perinuclear theca nor the fatty acid composition of sperm. Moreover, epididymal sperm numbers were not affected in FABP9^−/− mice. Therefore, we conclude that FABP9 plays only a minor role in providing the murine sperm head its characteristic shape and is not absolutely required for spermatogenesis or sperm function.     

Laboratory scale examination of the effects of overloading on the anaerobic digestion by glycerol

The anaerobic digestion of pure glycerol, which produces a baseline acetic acid to propionic acid ratio of 0.2, was studied in laboratory scale reactors (3 l working volume) at mesophilic temperature (37 °C) with 3000 mg chemical oxygen demand (COD) l⁻¹d⁻¹. During the experiment tVFA and C2-C6 VFA analysis and daily biogas yield measurement were carried out. Following 10 days of a 15% d⁻¹ increase in the organic loading rate (OLR) of 3.0-10.5 g COD l⁻¹d⁻¹, the concentration of propionic acid increased to 6200-8000 mg l⁻¹. Then the inoculum was divided into three parts feeding with 100% glycerol, 50% glycerol + 50% acetic acid, and 50% glycerol + 50% thick stillage, (presented in % of 2.60 g COD l⁻¹d⁻¹ OLR), respectively. The application of co-substrates reduced the recovery period by 5 days compared to feeding with pure glycerol. When the reactors were loaded with glycerol again (10% OLR raise per day) the previously applied co-substrates had a positive effect on the VFA composition and the biogas yield as well.     

Kinetics of complexin binding to the SNARE complex: correcting single molecule FRET measurements for hidden events

Virtually all measurements of biochemical kinetics have been derived from macroscopic measurements. Single-molecule methods can reveal the kinetic behavior of individual molecular complexes and thus have the potential to determine heterogeneous behaviors. Here we have used single-molecule fluorescence resonance energy transfer to determine the kinetics of binding of SNARE (soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor) complexes to complexin and to a peptide derived from the central SNARE binding region of complexin. A Markov model was developed to account for the presence of unlabeled competitor in such measurements. We find that complexin associates rapidly with SNARE complexes anchored in lipid bilayers with a rate constant of 7.0 × 10⁶ M⁻¹ s⁻¹ and dissociates slowly with a rate constant of 0.3 s⁻¹. The complexin peptide associates with SNARE complexes at a rate slower than that of full-length complexin (1.2 × 10⁶ M⁻¹ s⁻¹), and dissociates much more rapidly (rate constant >67 s⁻¹). Comparison of single-molecule fluorescence resonance energy transfer measurements made using several dye attachment sites illustrates that dye labeling of complexin can modify its rate of unbinding from SNAREs. These rate constants provide a quantitative framework for modeling of the cascade of reactions underlying exocytosis. In addition, our theoretical correction establishes a general approach for improving single-molecule measurements of intermolecular binding kinetics.     

Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution

Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 μl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral’s sperm.     

Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm

The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, ∼200 × 10⁶ sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300 × g for 20 min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean ± SD of 16.4 ± 8.7, 10.7 ± 8.9, and 2.3 ± 1.7%, respectively; P < 0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02 ± 0.01, 0.02 ± 0.04, 0.03 ± 0.04, and 0.44 ± 0.22 × 10⁶ cells/mL, respectively; P < 0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P > 0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P < 0.05), due to the lowest proportion of coiled tails (P < 0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P > 0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up.     

Flow cytometric sorting of human sperm: MicroSort clinical trial update

This report provides a summary of MicroSort^® efficacy in separation of X- from Y-chromosome bearing human sperm (XSort^® and YSort^®, respectively), clinical outcomes, and the sex of the resultant babies when sorted sperm were used for intrauterine insemination (IUI), in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Clinical trial participants were married couples seeking reduced X-linked genetic disorder risk or family balancing. Sperm were stained with Hoechst 33342, sorted by flow cytometry, then used or cryopreserved for subsequent use. Fluorescence in situ hybridization (FISH) analysis determined the post-sort enrichment (purity) for X- and Y-bearing sperm. Birth and pediatric records were evaluated for incidence of congenital malformations. Between June 1994 and January 2007, patients underwent 3629 IUI cycles, 1642 IVF/ICSI cycles with fresh embryo transfer (ET) and 99 frozen embryo transfer (FET) cycles after MicroSort^®. Of 5871 total sorts, 74.9% were XSort^® and 25.1% were YSort^®. IVF/ICSI fertilization rate was 70.7% and 93.8% of 2PN embryos cleaved. The pregnancy rates for IUI, IVF/ICSI, and FET were 15.6, 32.0, and 33.3%, respectively, while miscarriage rates were 15.7, 14.3, and 33.3%, respectively. Post-sort purity averaged 87.9% (XSort^®) and 73.4% (YSort^®). A total of 1125 clinical pregnancies yielded 943 babies born and 167 ongoing pregnancies. For babies born, XSort^® resulted in 92.0% females and YSort^® yielded 81.5% males. Postnatal follow-up showed a 2.6% major congenital malformation rate, with no recurrent pattern or clustering of malformations. FISH results confirmed MicroSort^® enrichment of X- and Y-bearing sperm populations that closely corresponded with the sex of the resultant child. Fertilization, cleavage, spontaneous abortion, and pregnancy rates as well as incidence of major congenital malformations were comparable to those in literature reports utilizing unsorted sperm.     

Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor

Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 × 10⁶ M⁻¹ s⁻¹ for k_a and 3.15 × 10⁻³ s⁻¹ for k_d. These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k_a, k_d, and K_D as 8.48 × 10⁵ M⁻¹ s⁻¹, 1.7 × 10⁻³ s⁻¹, and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction.     

The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36 h later. Sorted (S) and non-sorted (NS) doses of 1 or 15 × 10⁶ motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66 h after progestagen withdrawal. An additional treatment dose of 0.5 × 10⁶ S or NS sperm was inseminated at the 58 h time point (n = 60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1 × 10⁶ and 15 × 10⁶ sperm achieved similar pregnancy rates, regardless of sperm type, at 58 h (S1: 46 ± 9.4%; S15: 43 ± 9.3%; NS1: 41 ± 9.2%; NS15: 49 ± 9.4%). However, pregnancy rates were lower (P < 0.05) for doses of 1 than 15 × 10⁶ sperm inseminated at 50 (15 ± 6.3% vs. 36 ± 9.1%), 54 (14 ± 4.4% vs. 55 ± 7.3%), 62 (33 ± 6.9% vs. 54 ± 7.3%), and 66 h (29 ± 8.6% vs. 56 ± 9.5%). There was no difference between S and NS sperm for inseminations with 0.5 × 10⁶ motile sperm at 58 h after PR (15 ± 3.6% vs. 14 ± 3.3%), nor with 15 × 10⁶ motile sperm at all insemination times (49 ± 6.3% vs. 49 ± 6.3%). However, fertility was higher for S than NS sperm at the 1 × 10⁶ dose level (37 ± 6.1% and 16 ± 4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.     

Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils

The kinetics of Ca²⁺-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca²⁺] or the Ca²⁺-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca²⁺] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s⁻¹ and ∼330 s⁻¹, respectively. At low [Ca²⁺] (pCa 6.6), the slower rate was reduced to ∼25 s⁻¹, but was still ∼50-fold higher than the rate constant of Ca²⁺-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca²⁺] from pCa 5.0-7.9 induced a fluorescence decay with a rate constant of 39 s⁻¹, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca²⁺-binding (k_B ≈ 120 μM⁻¹ s⁻¹) and dissociation (k_D ≈ 550 s⁻¹); and 2), slower switch-on (k_on = 390s⁻¹) and switch-off (k_off = 36s⁻¹) kinetics. At high [Ca²⁺], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.