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J M Morrell

Publications and source records attributed to J M Morrell.

At least 19 recordsLinked to original sources

Update on semen technologies for animal breeding.

Despite the scale of the livestock breeding industry, where many millions of insemination doses are prepared each year, sperm preparation techniques are used infrequently in animal assisted reproduction compared with its human counterpart. However, some of the techniques used for human sperm preparation, for example, density gradient centrifugation, improve the quality of sperm preparations which is, in turn, reflected by an increased conception rate. The preparation technique separates motile spermatozoa with normal morphology and intact DNA from the total sperm population, leaving behind immature or senescent spermatozoa, morphologically abnormal ones and those with damaged DNA. Furthermore, the motile spermatozoa are removed from the seminal plasma which carries cells, cellular debris and reactive oxygen species, as well as pathogens. Gradient-prepared spermatozoa survive longer, either in liquid storage or when cryopreserved, and are free of bacteria and viral infectivity if prepared carefully. Preparation techniques such as density gradient centrifugation, or the simplified single layer centrifugation technique, have considerable potential for aiding sperm preparation from poor quality semen samples, such as may be obtained from unselected semen donors in captive breeding programmes, or from performance horses. Moreover, the removal of pathogens has important implications, both for disease control and for avoiding the use of antibiotics in semen extenders, which can be detrimental to sperm survival.

Animals↗

Effective removal of equine arteritis virus from stallion semen.

REASONS FOR PERFORMING STUDY: A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. OBJECTIVES: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. METHODS: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing technique. The sperm preparations were tested by PCR for the presence of EAV. The procedure was repeated using an ejaculate from a known shedding stallion, testing processed and unprocessed aliquots by PCR and virus isolation. RESULTS: Virus-free sperm preparations were obtained using the double sperm processing technique. The 'swim-up' step is apparently required to ensure complete virus removal. CONCLUSIONS: The double semen processing technique is potentially a useful and simple tool for the removal of EAV from the semen of shedding stallions. POTENTIAL RELEVANCE: The inclusion of density gradient centrifugation and 'swim-up' in protocols for the processing of semen for artificial insemination could help prevent the transmission of viral diseases carried in semen, such as EAV.

Animals↗

Effect of semen extender and density gradient centrifugation on the motility and fertility of turkey spermatozoa.

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.

Animals↗

Reduced senescence and retained nuclear DNA integrity in human spermatozoa prepared by density gradient centrifugation.

PURPOSE: To investigate whether removal of extraneous cells and immotile spermatozoa from a sperm preparation by density gradient centrifugation could help to maintain normal spermatozoa in a viable state and retain their deoxyribonucleic acid integrity. METHODS: Sperm motility was assessed on a daily basis in aliquots of neat semen, extended semen, and spermatozoa prepared on a PureSperm density gradient. At the same time, aliquots of each sperm sample were preserved for TUNEL assay and nick translation. RESULTS: Spermatozoa prepared using density gradient centrifugation survived three times as long as spermatozoa in neat semen or in extended semen. Both deoxyribonucleic acid integrity and sperm motility were retained in the gradient preparations. CONCLUSIONS: Preparing spermatozoa by density gradient centrifugation is advantageous in prolonging sperm survival and maintaining deoxyribonucleic acid integrity, presumably by removing sources of reactive oxygen species. Stored spermatozoa could be used for a second attempt at fertilization if oocyte immaturity was suspected.

Adult↗

An alternative to PVP for slowing sperm prior to ICSI.

BACKGROUND: There is a growing awareness of potential problems in exposing sperm to polyvinylpyrrolidone (PVP) to slow their motility, a procedure commonly used prior to ICSI. The study presented here evaluates an alternative product for slowing sperm motility, which contains hyaluronate, a substance found naturally in the reproductive tract. METHODS: Computerized sperm motility analysis was used to compare the motilities of sperm exposed to either a PVP-containing product (ICSI-100), or a hyaluronate-containing product (SpermCatch), or control sperm resuspended in a sperm maintenance medium. A subjective assessment was made of the ease with which sperm could be isolated and be drawn into, and expelled from, an injection pipette after having their tails nicked. Sperm exposed to either ICSI-100 or SpermCatch were used for ICSI. Fertilization rate, zygote development, grading, and outcome of transfer were recorded for the two treatment groups. RESULTS: The hyaluronate-containing product slowed sperm motility sufficiently for the sperm to be captured in an injection pipette, was easy to draw into and expel from the pipette, prevented sperm sticking to plastic or glassware, and did not affect post-injection zygote development. Clinical pregnancy rates were similar for the two groups. CONCLUSIONS: This product represents an alternative to PVP for slowing sperm motility prior to ICSI.

Cleavage Stage, Ovum↗

Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice.

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility. A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare. The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitro and in vivo screening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.

Animal Welfare↗

The role of maternal cognitions in infant sleep problems as assessed by a new instrument, the maternal cognitions about infant sleep questionnaire.

Sleeping problems in young children are very common, affecting up to 20% of 1-year-olds. Behaviour therapy is the most widely recognised treatment but a significant proportion of parents find it aversive. This suggests that parental cognitions may be important and underevaluated. The aims of this study were therefore fourfold: (1) to describe the development of a new tool to measure maternal cognitions in relation to infant sleeping problems, the Maternal Cognitions about Infant Sleep Questionnaire (MCISQ): (2) to assess the psychometric properties of the MCISQ; (3) to test the hypothesis that maternal cognitions will be associated with, and specific to, infant sleep problems; and (4) to discuss the implications of the findings with respect to aetiological models of infant sleep problems, treatment, and future research. Results showed that maternal cognitions about setting limits, anger at the infant's demands, and doubts about parenting competence were significantly associated with infant sleep problems, whereas cognitions about infant hunger or cot death were not. It is hypothesised that these cognitive responses, if extreme, may disrupt sensitive parent-child interactions, which over time could lead to impairments in the development of infant self-regulation.

Adult↗

Artificial insemination in Callithrix jacchus using fresh or cryopreserved sperm.

Assisted reproductive techniques are needed urgently to facilitate the captive breeding of many New World primate species which are endangered in the wild and to assist the effective genetic management of small colonies. A protocol was devised for artificial insemination in the common marmoset, Callithrix jacchus, using ejaculated sperm obtained by vaginal washing after copulation. A double insemination protocol was employed, with the first insemination taking place the day before ovulation was expected to occur and the second 48 h later. All six females inseminated with fresh ejaculated sperm became pregnant, delivering a total of 16 offspring at term. The gestation lengths and litter sizes were not statistically different from those observed in pregnancies following natural mating. The insemination protocol was adapted for use with cryopreserved ejaculated sperm by including an additional insemination on the day of expected ovulation, to take into account differences in the capacitation time of frozen-thawed sperm compared to fresh sperm. Three out of six females inseminated according to this triple insemination schedule, conceived, although one female subsequently resorbed twin foetuses approximately 100 days later. The remaining two pregnant females delivered four babies at term, one singleton and one set of triplets. In the final group, six females were inseminated with low doses of cryopreserved epididymal sperm using the same triple insemination protocol used for frozen-thawed ejaculated sperm. One female conceived, delivering triplets.

Animals↗

Cryopreservation of non-human primate sperm: priorities for future research.

Wild populations of many non-human primate species have declined alarmingly due to habitat destruction, hunting and genetic isolation. Captive breeding programmes to aid species survival could be enhanced by the use of assisted reproductive techniques, such as artificial insemination (AI), if a source of viable sperm was readily accessible. Cryobanks of primate sperm could provide such a supply if techniques for freezing sperm could be developed. Although sporadic attempts to cryopreserve primate sperm have been reported for some of the more frequently encountered zoo-maintained species, there is limited information available on techniques for sperm collection and storage. It is vital that adequate reporting of all cryopreservation attempts be made to avoid repetition of inappropriate methodologies and wastage of valuable genetic material from rare or endangered animals. An integrated approach to the cryobanking of non-human primate sperm is considered to be essential for species conservation. In this review, the factors affecting the success of sperm cryopreservation are outlined, existing information is compiled from previous reported attempts at cryopreservation, and suggestions are made for cryopreserving sperm in further non-human primate species. Moreover, recommendations are given for additional studies to augment existing data. It is intended that this information should serve as a guide for developing cryopreservation protocols in the future, particularly for endangered species.

Animals↗

Birth of offspring following artificial insemination in the common marmoset, Callithrix jacchus.

The objective of the study was to develop a method for artificial insemination (AI) in the common marmoset, a New World primate species. For AI to be successful, sperm must be deposited at an appropriate site and time in the female reproductive tract, details of which are currently not available for Callitrichid species. Epididymal sperm were deposited in the cervix of 18 marmoset monkeys (Callithrix jacchus) around the time of expected ovulation using either 3, 2, or 1 inseminations. Six out of 18 females conceived, resulting in the first reported births following AI in this species. These pregnancies show that the presence of coagulum in the vagina and the stimulus of the female reproductive tract by natural mating are not essential for effective sperm transport in this species. Although 3 different timing regimes for sperm deposition relative to ovulation were employed, no protocol was demonstrably better than the others in terms of number of conceptions. The proportions of motile, live, and morphologically normal sperm in the suspensions used for AI were comparable with published values for ejaculates from fertile male macaques. These preliminary results indicate that births are possible following AI in marmosets: the technique could be used to aid effective genetic management of the species and possibly to facilitate captive breeding of endangered Callitrichids.

Animals↗

Fertilization-promoting peptide in reproductive tissues and semen of the male marmoset (Callithrix jacchus).

Fertilization-promoting peptide (FPP) is present in the prostate gland and semen of some mammals, and has been shown to enhance the fertilizing ability of both epididymal mouse and ejaculated human spermatozoa. The novel peptide may prove of importance for the treatment of some cases of male infertility, and a suitable animal model would be useful to test this hypothesis. To this end, we examined reproductive tissues and semen of the male marmoset for the presence of FPP. Peptides were extracted from seminal plasma, testes, prostate, and bulbourethral glands of intact and castrated male marmosets. The peptides were identified by ion-exchange chromatography followed by radioimmunoassay. The mean concentration of FPP immunoreactivity in semen from intact males was 58.7 nM (SE +/- 9.9 nM, n = 10), and anion-exchange chromatography revealed FPP as the only immunoreactive peptide present. Analysis of tissues revealed that FPP in semen was likely to be derived from the prostate gland, which contained this peptide as the major source of immunoreactivity (10.86 pmol/gland; SE +/- 4.39 pmol/gland, n = 4). Only low concentrations of FPP were detectable in the bulbourethral glands, and the peptide was undetectable in the testis. Surprisingly, FPP was readily detectable in the seminal plasma from one castrated marmoset and was present in the prostate gland from 3 castrates at levels which did not differ significantly from those in intact animals (5.47 pmol/gland, SE +/- 1.64 pmol/gland, n = 3). Plasma testosterone measurements indicated that residual circulatory androgens remained after castration, which may be consistent both with the maintenance of mating behavior and the presence of prostatic FPP. We conclude that FPP is present within the prostate gland and seminal plasma of the marmoset at concentrations consistent with a role in male fertility in this species.

Animals↗

CASA as an aid to selecting sperm suspensions for artificial insemination in Callithrix jacchus.

The ability to use sperm motility parameters, obtained by computer-assisted sperm motility analysis (CASA), as an aid to selecting sperm samples for artificial insemination (AI) would have considerable benefits for commercial organizations and for the captive breeding of endangered species. In this study the Hobson sperm tracker (HST) was validated for use with spermatozoa from Callithrix jacchus, the common marmoset, by comparing values for straight line velocity by CASA with those obtained by direct measurement of sperm tracks. Using the settings established during validation, ejaculated and epididymal spermatozoa were analysed with the HST. The range of values for velocity parameters were used to establish expected motility profiles for the two types of spermatozoa as follows: for epididymal spermatozoa (concentration 2.2 - 85.8 x 10(6) spermatozoa/mL) VCL 109.8-155.9, VSL 75.2-141.5, VAP 85.8-142.1, MAD 13.7-40.7, ALH 3.8-7.8, BCF 1.4-4.2, LIN 40.5-91.1% and STR 70.1-97.1%; for ejaculated spermatozoa (concentration 3.2-82.0 x 10(6) spermatozoa/mL) VCL 89.6-136.7, VSL 69.6-110.3, VAP 74.5-121.9, MAD 19.2-29.3, ALH 3.0-9.9, BCF 2.8-5.5., LIN 65.4-85.3% and STR 93.8-97.7%. Epididymal spermatozoa from males which were not sexually active had significantly lower values for VCL, VSL and VAP, while values for MAD were significantly higher than for spermatozoa from sexually active males (p < 0.031). Sperm concentration affected motility parameters significantly. Although motility parameters differed according to the batch of medium used, the differences were not statistically significant. Epididymal sperm samples had significantly higher VCL, VSL and VAP but lower BCF and LIN than ejaculated sperm samples of the same concentration diluted in the same batch of medium, while MAD, ALH and STR were not different. Urine contamination significantly reduced VCL, VSL and VAP (p < 0.008, < 0.016 and < 0.008, respectively, sample size = 7) whereas MAD, ALH, BCF, LIN and STR were not affected. Therefore CASA could be useful in screening ejaculates for use in Al to eliminate samples with unusual motility patterns.

Animals↗

Maturation of sperm motility in the epididymis of the common marmoset (Callithrix jacchus) and the cynomolgus monkey (Macaca fascicularis).

The acquisition by spermatozoa of the capacity for movement upon liberation from the epididymal duct into buffer is one of the obvious changes associated with sperm maturation and, hence, studies of sperm kinematics might provide good indices for sperm maturation. Profiles of development in the movement pattern of spermatozoa taken from six regions of the epididymis were established in marmoset and cynomolgus monkeys using computer-assisted sperm analysis. Motion of spermatozoa from initial regions was sluggish and irregular, then developed in the more distal region into erratic movement effected by an increase in the vigour of flagellation as reflected by curvilinear velocity. The tendency of immature spermatozoa to display circular motion was more common in the marmoset than in the cynomolgus monkey. In the marmoset the most drastic changes in percentage motility and kinematic parameters of spermatozoa occurred between the distal caput and the proximal corpus; maximal values for these parameters were found around the distal corpus epididymis. For the cynomolgus spermatozoa the sharpest gain in percentage motility was also achieved between the caput and proximal corpus epididymis, but with full kinematic development in the proximal cauda epididymis. These maturational changes were not synchronous among spermatozoa from the same site as they moved along the epididymis, as reflected in the heterogeneity of each sample in the kinematic properties of individual spermatozoa, especially those from the proximal corpus epididymis. The maturational profiles in the monkeys are comparable to the situation in the human, for which they may provide a useful model.

Animals↗

Collection of semen from marmoset monkeys (Callithrix jacchus) for experimental use by vaginal washing.

Assisted reproductive techniques make an important contribution to the conservation of endangered primate species. In our laboratories marmoset monkeys (Callithrix jacchus) are used as a model species for developing assisted reproductive technologies for New World primates. The studies require a reliable method for collecting functional sperm from these small animals. For this purpose a minimally invasive procedure, vaginal washing after natural mating, was evaluated for its suitability as a routine method of obtaining ejaculated sperm of high quality. The objective of the first series of tests was to identify a behavioural pattern which was easily discernible by the observer and provided a reliable indication that ejaculation had occurred. In a second series of tests the influence of length of separation of the male prior to copulation on the quality of the ejaculate was evaluated. Six adult ovariectomized and five adult intact females were used for vaginal washing, while their mates served as donors of ejaculates. Matings at specific times were achieved by separating the males from their females for a certain time period and subsequently introducing either their own mates or unfamiliar males to the females. After each observed mating vaginal washing was performed on the unsedated females. The seminal samples obtained were analysed for sperm concentration, the proportion of motile sperm and the proportion of live sperm. The results have shown (a) that ejaculation was indicated with a reliability of 86.1% by the behaviour of the female, who terminates a copulation by moving away from her partner; (b) that separation of the male for up to six days had no negative effect on the sperm quality; (c) that the sperm samples were of high quality. Vaginal washing after natural matings appears to be a practical, reliable and gentle method for routinely collecting ejaculates from marmoset monkeys and possibly other New World primates.

Animals↗

Preliminary investigation of an ELISA kit as a qualitative assay for rabbit progesterone.

An ELISA kit for measuring plasma progesterone in bitches was investigated to determine its potential as an assay for rabbit progesterone. The results suggest that the kit can be used for the qualitative assessment of rabbit progesterone, either in plasma or whole blood. Pregnant and pseudopregnant rabbits can be differentiated accurately at day 19 which is reported to be the optimum time to remate pseudopregnant females. The fact that whole blood can be used instead of plasma offers considerable potential benefit to rabbit breeders.

Animals↗

Analyses of DNA content of living spermatozoa using flow cytometry techniques.

A two-dimensional fluorescence analysis of spermatozoa stained with Hoechst 33342 was carried out using an Epics V flow cytometer. This analysis involved the measurement of fluorescence both in the conventional manner at 90 degrees and at a narrow forward angle (nominally 0 degree) to the direction of the interrogating laser beam. The arrangement provided an optical means of distinguishing the orientation of flattened particles passing through the interrogation system and enabled the verification and reinterpretation of one-dimensional (90 degree) fluorescence histogram data obtained previously. We now consider that the distinction between live and dead spermatozoa was over simplified and that the observed differences are due partly to an artefact of orientation. It has been confirmed that our earlier use of the low fluorescence peak, consisting of (bull, rabbit, sheep and pig) spermatozoa passing through the interrogation point obliquely to both detectors, as criterion for sorting X- from Y-bearing spermatozoa, is more practical than the use of a very much smaller subpopulation, based on limits imposed by selecting only spermatozoa accurately orientated with their narrow edges to one detector, while measurement of total fluorescence (DNA content) was made simultaneously from a broad face by the other detector. We report marked differences in the rate of staining and in attainment of a staining equilibrium between spermatozoa from different animals or from different ejaculates collected on the same or different days from the same animal. This variation introduces an element of subjectivity into the use of flow cytometry for sorting X- and Y-spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

TRH-related peptides in the rabbit prostate complex during development.

The novel peptide, pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2), has recently been isolated and characterized from the rabbit prostate complex. The tripeptide is present in high concentrations in the prostate complex and semen, together with a 40-50 residue polypeptide which contains a TRH-immunoreactive fragment at its C-terminus. The present study investigates changes in the levels of these TRH-related peptides in rabbits aged 11 weeks, 4 months, 7 months, 13 months and 2 years. For each age group the peptides were extracted from the prostate complex, separated by gel exclusion chromatography, and located by TRH radioimmunoassay. The TRH-immunoreactive fragment was released from the polypeptide by trypsin digestion prior to radioimmunoassay. Very low concentrations of TRH-immunoreactive peptides were present at 11 weeks of age, but considerable levels of both peptides were found in all the other age groups. Anion exchange chromatography, under conditions which resolve TRH and pGlu-Glu-ProNH2, showed that the majority of the low molecular weight TRH immunoreactivity co-eluted with synthetic pGlu-Glu-ProNH2. The remaining TRH immunoreactivity, which had not bound to the anion resin, also failed to bind to a cation exchange column at pH 2.0, indicating that it was not authentic TRH. Dissection of the prostate complex into its four constitutive regions (vesicular gland, coagulating gland, prostate and bulbourethral gland) followed by extraction, chromatography and TRH radioimmunoassay of each region showed that the TRH-related peptides were located in the prostate.

Aging↗