Birth of Koalas Phascolarctos cinereus at Lone Pine Koala Sanctuary following artificial insemination

This paper documents the successful development of an artificial insemination (AI) programme for the Koala Phascolurctos cinereus. The protocols for trials involving two methods to induce ovulation and two insemination techniques are described. In Trial 1, interrupted coitus using a 'teaser'♂ successfully induced ovulation in nine Koalas. Five ♀♀ were inseminated while conscious using a modified 'foley catheter' (Cook insemination catheter) resulting in the births of two offspring. The other four ♀♀ were anaesthetized and inseminated using a technique which allowed visualization of the most cranial portion of the urogenital sinus, where semen was deposited using a 3.5 Fr. 'Tom-cat catheter' (urogen-itoscopic insemination). Three of the four ♀♀ inseminated by this technique produced pouch young. Microsatellite analysis of DNA from the pouch young excluded the teaser ♀♀ as possible sires, confirming that all offspring were sired by donor sperm. In Trial 2, eight ♀♀ were induced to ovulate by injecting them with 250 International Units of human chorionic gonadotrophin (hCG). A luteal phase was confirmed in all eight ♀♀ but only one gave birth following urogenitoscopic insemination. The Koala pouch young in this study are the first of any marsupial to be conceived and born following A1 procedures. Details of the A1 procedures used are presented and the significance of A1 to the conservation biology of P. cinereus discussed.

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.

Synchronization of estrus and fertility in zebu beef heifers treated with three estrus synchronization protocols

The effects on estrus and fertility of 3 estrus synchronization protocols were studied in Brahman beef heifers. In Treatment 1 (PGF protocol; n=234), heifers received 7.5 mg, im prostianol on Day 0 and were inseminated after observed estrus until Day 5. Treatment 2 (10-d NOR protocol; n=220) consisted of norgestomet (NOR; 3 mg, sc implant and 3 mg, im) and estradiol valerate (5 mg, im) treatment on Day -10, NOR implant removal and 400 IU, im PMSG on Day 0, and AI after observed estrus through to Day 5. Treatment 3 (14-d NOR+PGF protocol; n=168) constituted a NOR implant (3 mg, sc) on Day -14, NOR implant removal on Day 0, PGF on Day 16, and AI after observed estrus through to Day 21. All heifers were examined for return to estrus at the next cycle and inseminated after observed estrus. The heifers were then exposed to bulls for at least 21 d. During the period of estrus observation (5 d) after treatment, those heifers treated with the PGF protocol had a lower (P

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.

Embryonic mortality in buffaloes synchronized and mated by AI during the seasonal decline in reproductive function

The aim was to determine the factors that contribute to embryonic mortality in buffaloes mated by AI during a period of increasing day length which corresponds to a natural decline in reproductive activity. Italian Mediterranean buffalo cows (n = 243) showing regular estrous cycles were synchronized using the Ovsynch-TAI program and mated by AI at 16 and 40 h after the second injection of GnRH. Blood samples were collected on Days 10 and 20 after the first AI and assayed for progesterone (P-4). Pregnancy diagnosis was undertaken on Days 26 and 40 after the first AI using rectal ultrasonography. Buffaloes with a conceptus on Day 26 but not on Day 40 were judged to have undergone embryonic mortality and for these animals uterine fluid was recovered by flushing and analysed for common infectious agents. Estrus synchronization was achieved in 86% of buffaloes and the pregnancy rate on Day 40 was 34%. Embryonic mortality between Days 26 and 40 occurred in 45% of buffaloes and was associated with the presence of significant infectious agents in only 10 buffaloes (8%). Concentrations of P-4 on Day 10 after AI were higher (P < 0.05) in buffaloes that established a pregnancy than in buffaloes that showed embryonic mortality that was not associated with infectious agents. Similarly, on Day 20 after AI P-4 concentrations were higher (P < 0.01) in pregnant buffaloes compared with non-pregnant buffaloes and buffaloes that had embryonic mortality. It is concluded that a reduced capacity for P-4 secretion can explain around 50% of embryonic mortalities in buffaloes synchronised and mated by AI during a period of low reproductive activity and that other as yet unidentified factors also have a significant effect on embryonic survival. (c) 2004 Elsevier Inc. All rights reserved.

Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.

Fixed bed reactors with catalyst poisoning - first order kinetics.

The long performance of an isothermal fixed bed reactor undergoing catalyst poisoning is theoretically analyzed using the dispersion model. First order reaction with dth order deactivation is assumed and the model equations are solved by matched asymptotic expansions for large Peclet number. Simple closed-form solutions, uniformly valid in time, are obtained.

A demonstration of artificial dissipation effects in some finite volume solution methods for the Navier-Stokes equations

The artificial dissipation effects in some solutions obtained with a Navier-Stokes flow solver are demonstrated. The solvers were used to calculate the flow of an artificially dissipative fluid, which is a fluid having dissipative properties which arise entirely from the solution method itself. This was done by setting the viscosity and heat conduction coefficients in the Navier-Stokes solvers to zero everywhere inside the flow, while at the same time applying the usual no-slip and thermal conducting boundary conditions at solid boundaries. An artificially dissipative flow solution is found where the dissipation depends entirely on the solver itself. If the difference between the solutions obtained with the viscosity and thermal conductivity set to zero and their correct values is small, it is clear that the artificial dissipation is dominating and the solutions are unreliable.