Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

Hebrew University of Jerusalem

Essential oils are used in foods, cosmetics, and pharmaceuticals. Despite the recent marketing of novel essential-oil-containing patches, there is no information on their production, constituents, or physical properties. The objectives of this study were to produce essential-oil patches and characterize their physical properties. The essential oil of Lavandula angustifolia (lavender) was included at concentrations of 2.5% to 10% in patches manufactured from the exudate gum karaya, propylene glycol, glycerol, emulsifier, and optionally, potato starch as filler. Inclusion of essential oil reduced patch strength, stiffness, and elasticity relative to patches without essential oil. Inclusion of starch in the essential-oil patches strengthened them, but reduced their elasticity. Patches' adhesion to substrate was examined by both peeling and probe-tack tests: the higher the inclusion of essential oils within the patch, the larger the decrease in its adhesion to substrate. Addition of starch to essential-oil-containing patches increased their adhesion relative to their essential-oil-only counterparts. Scanning electron micrographs of the patches provided evidence of entrapped starch granules. Although inclusion of essential oil reduced both the mechanical properties and adhesion of the patches, a high proportion of essential oil can still be included without losing patch integrity or eliminating its adhesiveness to the skin.

Physical properties of gum karaya-starch-essential oil patches.

Incorporation rates of dietary omega-3 (n-3) fatty acids (FAs) from different sources into bull plasma and sperm and the effects on physiological characteristics of fresh and frozen-thawed semen were determined. Fifteen fertile bulls were assigned to three treatment groups and supplemented for 13 weeks with encapsulated fat: (1) SFA-360 g/d per bull saturated FA; (2) FLX-450 g/d per bull providing 84.2 g/d C18:3n-3 (α-linolenic acid) from flaxseed oil; and (3) FO-450 g/d per bull providing 8.7 g/d C20:5n-3 (eicosapentaenoic acid) and 6.5 g/d C22:6n-3 (docosahexaenoic acid, DHA) from fish oil. Blood samples were taken every 2 weeks and semen was collected weekly. With respect to the FA supplements, the proportion of α-linolenic acid in plasma increased in the FLX bulls, whereas that of DHA was increased in the FO bulls, within 2 weeks. However, changes in the sperm FA fraction were first expressed in the sixth week of supplementation: in the FO and FLX bulls the DHA proportion increased (P < 0.001), whereas that of C22:5n-6 FAs (docosapentaenoic acid [DPA] n-6) decreased (P < 0.001). Sperm motility and progressive motility in fresh semen were higher (P < 0.05), and the fading rate tended to be lower in the FLX than in FO bulls (P < 0.06). Furthermore, sperm motility, progressive motility, and velocity in frozen-thawed semen were higher in FLX than in the other groups (P < 0.008). These findings indicate that the proportion of DHA in sperm can be increased at the expense of DPAn-6 by either FO or FLX supplementation, indicating de novo elongation and desaturation of short- into longer-chain n-3 FAs in testes. Furthermore, the moderate exchange of DHA and DPAn-6 in the FLX group's sperm was associated with changes in the characteristics of both fresh and frozen-thawed semen, suggesting the importance of the ratio between these two FAs for sperm structure and function.

Dietary α-linolenic acid from flaxseed oil or eicosapentaenoic and docosahexaenoic acids from fish oil differentially alter fatty acid composition and characteristics of fresh and frozen-thawed bull semen.

Ovulation failure, follicular persistence, and formation of follicular cysts are known to impair dairy cow fertility. Although the underlying mechanism is not entirely clear, stress-induced alteration in adrenal hormone secretion can cause these ovarian pathologies. Six synchronized lactating cows were scanned daily by ultrasound, and plasma samples were taken throughout the estrous cycle. Treatment cows (n = 3) were administered with ACTH analog every 12 h from day 15 to day 21 of the cycle to induce formation of follicular cysts. Ovaries were collected at the slaughterhouse on day 23 of the cycle before appearance of follicular pathologies. Control cows (n = 3) were administered placebo, resynchronized, and administered PGF2α on day 6 of the new cycle to induce development of a preovulatory follicle. Follicular fluid was aspirated from the preovulatory follicles of each group to determine their steroid milieu. Slices were taken from the follicular wall for total messenger (m) RNA isolation and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Administration of ACTH increased (P < 0.02) plasma cortisol concentration and reduced (P < 0.01) milk production. Androstenedione and estradiol concentrations in the follicular fluids were lower (P < 0.05) in ACTH-treated follicles than those in controls. The mRNA expression of luteinizing hormone receptor, 3β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase (P450arom), and cytochrome P450 17α-hydroxylase (P450c17) were lower (P < 0.02) in the ACTH-treated vs control cows. On the other hand, the expression of steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage did not differ between groups. In addition, mRNA expression of vascular endothelial growth factor (VEGF)120 and VEGF164 was higher (P < 0.01) in control than in ACTH-treated follicles, but that for angiopoietin-1 and 2 did not differ between groups. Findings indicated that ACTH administration throughout preovulatory follicle development alters follicular steroidogenesis in association with impaired angiogenesis. Such alterations might explain, in part, the mechanism underlying ovulation failure and the formation of persistent or cystic follicles under stress. 

ACTH administration during formation of preovulatory follicles impairs steroidogenesis and angiogenesis in association with ovulation failure in lactating cows.

Reduced reproductive performance of lactating cows is strongly associated with environmental and pathogenic stressors. This review summarizes the most recent knowledge on the effects of acute or chronic heat stress (HS) and acute or chronic intramammary infection (IMI) on ovarian function. It also offers various approaches for improving the fertility of cows under chronic HS or IMI. Comparing the 2 stressors reveals a few similarities in the mode of alteration in the hypothalamus-pituitary-ovarian axis, in particular, in the follicle and its enclosed oocyte. Both HS and IMI cause a reduction in the preovulatory LH surge, with a pronounced effect in cows with IMI, and consequently, ovulation is being delayed or inhibited. Both stresses induce changes in follicular growth dynamics, reduce follicular steroidogenesis, and disrupt follicular dominance. Unlike their effects on follicular function, the effects of mastitis and HS on corpus luteum (CL) function are debatable. Under chronic summer thermal stress, several, but not all, studies show reduced progesterone secretion by the CL. Subclinical mastitis does not affect CL function, whereas the effect of clinical mastitis is controversial; some show a reduction in progesterone, whereas others do not. Both stresses have been found to impair cytoplasmic and nuclear maturation of oocytes, associated with reduced embryonic development. These findings have provided insights into the mechanism by which HS and IMI compromise fertility, which enable developing new strategies to mitigate these effects. For instance, treatment with GnRH and PGF2α to induce follicular turnover successfully improved conception rate in subpopulations of HS cows during the summer, in particular, primiparous cows and cows with high BCS. The "Ovsynch" program, also based on the use of GnRH and PGF2α, has been shown to improve conception rate of subclinical mastitic cows, most likely due to better synchronization of timing of ovulation with that of AI. Supplementing progesterone after AI improves conception rate of HS cows, particularly those with postpartum uterine disease and low BCS. It should be noted that similarities between the 2 stressors do not necessarily suggest a shared mechanism. Although not clear enough, an additive deleterious effects of HS and IMI on reproduction is suggested.

Comparing the effects of heat stress and mastitis on ovarian function in lactating cows: basic and applied aspects.

Among the components of the female reproductive tract, the ovarian pool of follicles and their enclosed oocytes are highly sensitive to hyperthermia. Heat-induced alterations in small antral follicles can be expressed later as compromised maturation and developmental capacity of the ovulating oocyte. This review summarizes the most up-to-date information on the effects of heat stress on the oocyte with an emphasis on unclear points and open questions, some of which might involve new research directions, for instance, whether preantral follicles are heat resistant. The review focuses on the follicle-enclosed oocytes, provides new insights into the cellular and molecular responses of the oocyte to elevated temperature, points out the role of the follicle microenvironment, and discusses some mechanisms that might underlie oocyte impairment. Mechanisms include nuclear and cytoplasmic maturation, mitochondrial function, apoptotic pathways, and oxidative stress. Understanding the mechanism by which heat stress compromises fertility might enable development of new strategies to mitigate its effects.

Effect of Heat Stress on Reproduction in Dairy Cows: Insights into the Cellular and Molecular Responses of the Oocyte.

Stress can affect reproductive performance of lactating cows by targeting the ovarian pool of follicles and their enclosed oocytes. Among the documented stressors are heat stress (i.e., high temperature-humidity index) as well as environmental and food toxins. Oocytes collected during the hot season are of lower quality than those collected in the winter, expressed by reduced oocyte maturation and developmental competence. A similar pattern has been reported for oocytes exposed to endocrine-disrupting chemicals. Whereas the underlying mechanism might differ among stressors, accumulating evidence suggests that stress-induced impairment of oocyte developmental competence involves alterations in mitochondrial functioning. Within the oocyte, mitochondria are involved in ATP generation, calcium homeostasis, regulation of cytoplasmic reduction-oxidation, signal transduction, and apoptosis. Summer heat stress is strongly associated with alterations in mitochondrial distribution and alterations in mitochondria membrane potential. Heat stress impairs the expression of mitochondrion-associated genes, in particular those related to mitochondrial DNA transcription and replication and encoding oxidative phosphorylation complexes for ATP production. Reduction of ATP levels below the required threshold is suggested to compromise the progression of oocyte maturation and, subsequently, embryonic development. Another mechanism associated with mitochondrial function is the increase in reactive oxygen species (ROS), which has been documented in oocytes exposed to heat stress or environmental toxicants. Oxidative phosphorylation in mitochondria is the major source of ROS. Under physiological conditions, ROS are essential for nuclear maturation; however, disequilibrium between ROS production and antioxidative capacity might lead to DNA damage and apoptosis. The current review provides new insights into the oocyte's cellular and molecular responses to stress with an emphasis on the mitochondria. It discusses some strategies to mitigate the effects of stress on the mitochondria, such as incorporation of coenzyme Q10-a key component of the mitochondrial respiratory chain-administration of antioxidants, and injection of healthy mitochondria. Exploring the oocyte's cellular and molecular responses, in particular that of the mitochondria, might lead to the development of new strategies to mitigate the effects of various stressors on fertility.

Symposium review: Reduction in oocyte developmental competence by stress is associated with alterations in mitochondrial function.

The oocyte achieves its developmental competence through the lengthy process of folliculogenesis. It can therefore potentially be exposed to various stressors while enclosed in the follicle. Oocyte maturation relies mainly on maternal sources. These include nuclear, cytoplasmic, and molecular maturation, which involve DNA and RNA organization. Maternal transcripts are dominant through the first embryonic cleavages, up until embryonic genome activation. Thus, it is suggested that any perturbations during oocyte storage, in particular of the maternal transcripts, might lead to genetic and/or epigenetic changes, which might be further expressed in the developing embryo. The review discusses the effects of three representative stressors-environmental heat stress, endocrine-disrupting compounds (phthalates), and inflammatory stress (mastitis)-shown to be involved in reduced fertility. The review highlights the carryover response from the oocyte to the developing embryo; it includes intracellular and molecular disruptive mechanisms with an emphasis on maternal transcripts. The review provides insights into the oocyte's cellular and molecular responses with an emphasis on the effects of various stressors on the maternal (nuclear and mitochondrial) transcripts and the association with embryonic development. A comparison between stressors might clarify, at least in part, a few open questions. For instance, (a) whether stress-induced alterations share the same mechanism and if so (b) whether this mechanism involves alterations of maternal transcripts; (c) whether stress-induced alterations in the maternal transcript are further expressed at the developing blastocyst stage, that is, after embryonic genome activation.

Zvi Roth

Hebrew University of Jerusalem

Fluorimetric Techniques for the Assessment of Sperm Membranes