Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires

This study determined the relationship between two measures of field fertility of I I high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability. The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2 h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 20001 was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and cCR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with cCR and with cNRR. The sperm parameters identified as being most predictive of cCR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2 h incubation at 37 degreesC (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR (P = 0.1) and cNRR (P = 0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group. (C) 2003 Elsevier B.V. All rights reserved.

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 mu M), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility. (c) 2006 Elsevier Inc. All rights reserved.

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Triscitrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P< 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa ( P< 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol ( P< 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.

Successful artificial insemination (AI) in the koala using neat and extended semen collected by electroejaculation (EE)

Presently AI in the koala has been based on the insemination of fresh undiluted semen collected with an artificial vagina (1). While this approach has been extremely successful, further refinement and implementation of AI for use with cryopreserved semen will require protocols that incorporate diluted semen collected by EE. Recent studies have shown that koala semen is likely to have an "ovulation factor" such that over-dilution may result in ovulation failure (2). The current study determined whether AI of EEed neat and/or diluted semen was capable of inducing a luteal phase and/or resulted in the production of pouch young. All koalas were inseminated in the breeding season between day 2 and 5 of oestrus and subsequently monitored for evidence of parturition (day 35) and return of oestrus. Successful induction of a luteal phase was based on evidence of an elevated progesterone concentration 28 days after insemination (2). All semen samples were collected by EE and seminal characteristics recorded (3). The diluent used for semen extension was Tris-citrate glucose (TCG) which contained antibiotics but no egg yolk (4). AI was conducted on conscious koalas using a "Cook koala insemination catheter" and a glass rod used to mimic penile thrusting (1). Three insemination treatments were used; (A) 1mL of undiluted semen (n = 9); (B) 2mL of 1:1 diluted semen (n = 9); and (C) 1 mL of 1:1 diluted semen (n = 9). The results of the AI trial are shown in Table 1. This study has shown that it is possible to use both neat and diluted semen (1:1; 1 or 2 mL) to successfully produce koala offspring at conception rates similar to those achieved following natural mating. Interestingly, dilution of semen had no apparent detrimental effect on induction of a luteal phase following AI.