Optical manipulation of microparticles and cells on silicon nitride waveguides.

We demonstrate the optical manipulation of cells and dielectric particles on the surface of silicon nitride waveguides. Glass particles with 2microm diameter are propelled at velocities of 15microm/s with a guided power of 20mW. This is approximately 20 times more efficient than previously reported, and permits to use this device on low refractive index objects such as cells. Red blood cells and yeast cells can be trapped on the waveguide and pushed along it by the action of optical forces. This kind of system can easily be combined with various integrated optical structures and opens the way to the development of new microsystems for cell sorting applications.

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses.

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.

[Father-male offspring transmission of seasonal variations in testicular diameter and percentage of abnormal sperm in the Ile-de-France ram. 1. Male offspring born in February]. / Etude de la transmission père-fils des variations saisonnières du diamètre testiculaire et du pourcentage de spermatozoïdes anormaux chez le bélier ile-de-France. 1. Fils nés en février.

An experiment was conducted on the Ile-de-France (IF) breed to determine if the more or less important sensitivity of the ram to photoperiodism came under genetic control. Five base breed unrelated rams (greater than or equal to 4 yr old), were chosen for this study: 2 good (I and II), 2 bad (III and IV), and an intermediate sire (V). Ram I, which died a few years before the experiment began, was selected on both the low amplitude of its sperm production during a 6-month period of artificial lighting and the very high fertilizing capacity of its sperm in spring. Rams II to V were controlled for 12 (percentage of morphologically abnormal spermatozoa, AM) or 14 (maximum antero-posterior scrotal diameter of both testes, DT) consecutive months. Following this period of control, seasonal variation in rams was assessed as follows: low (ram II), high (rams III and IV) and intermediate (ram V) seasonal variation rams. Breeding (artificial insemination) of these 5 rams to IF ewes resulted in 18 male offspring born in February and distributed as follows: rams I to III: 4 male offspring/ram (families 1 to 3, F1 to F3), rams IV and V: 3 male offspring/ram (families 4 and 5, F4 and F5). The 18 animals were controlled once a week (DT and AM) from 8, 5 to 46 months of age (7 periods, P1 to P7). Regarding DT, mean DT was higher in F1 and F2 than those of the whole population (WP) (represented by a discontinuous line of ordinate 5.0 in Graph 3), but F2 was closer to this population than F1. On the other hand, they were lower in the rams of F3 and F4. Differences between F3 or F4 and WP were lower in autumn than in spring. All families showed significant differences during the experimental periods except at P7 for pairs 3-5 and 4-5. Distances between F1, F2, F3 were always different (P less than 0.01 or P less than 0.001), whatever the size of the population (n = 3 or 4). Weekly F5 values varied in an opposite way to those of F3 and F4: increase in spring and decrease in autumn. Regarding AM, families did not differ as much as in DT (graph 4). Weekly variations in AM were also stronger. However, mean AM was almost always lower in F1 males and somewhat higher in F3 males to that of WP.(ABSTRACT TRUNCATED AT 400 WORDS)

Photoperiodic and melatonin treatments for the control of seasonal reproduction in sheep and goats.

The control of seasonal reproductive activity in sheep and goats in open sheds, needs extra-light (E) during the photosensitive phase (equivalent to long days, LD), followed by treatment with melatonin (equivalent to short days, SD). In autumn-born Ile-de-France or Lacaune ram lambs, 2 months of E followed by decreasing daylength for 90 days, advanced onset of the first breeding season by allowing males to reach their maximum testis volume and sperm production earlier than for untreated ram lambs. Substitution of decreasing daylength with melatonin implants allowed a transient increase in testis volume. Adult Ile-de-France rams maintained under short light rhythms with 2 month-period, demonstrated, during at least 2 consecutive years, a testis volume equivalent to that observed during the normal breeding season. These light-treated rams produced, during non-breeding season, spermatozoa in the same quantity and quality as during the normal breeding season. In anovulatory out-of-season dairy goats, E treatment was demonstrated to be necessary before melatonin treatment and melatonin to be necessary after E treatment to stimulate oestrous and ovulatory activities. Stimulation of the anovulatory females by the introduction of treated males ("male effect"), appeared to be necessary to obtain maximum stimulation of the treated females. Two months of E, followed by melatonin treatment (daily injection or drenching or subcutaneous implants) allowed cycles with ovulation to be maintained for more than 2 months. Although effective for control of out-of-season reproductive activity, melatonin slightly decreased milk production when applied soon after kidding. So, total control of reproduction in sheep and goats by manipulation of photoperiod in open sheds and melatonin treatments appears feasible in both sexes.

[Early prediction of the magnitude of seasonal variations in testicular diameter and percentage of abnormal spermatozoa in Ile-de-France rams. 1. Animals born in February]. / Recherche d'une prévision précoce de l'amplitude des variations saisonnières du diamètre testiculaire et du pourcentage de spermatozoïdes anormaux chez le bélier Ile-de-France. 1. Animaux nés en février.

The aim of this study, conducted in spring born animals, was to analyse if within a group of males of the same age, there is any relationship between the testis diameters (TD) or between the percentages of morphologically abnormal spermatozoa (AS) recorded in the same animal at two periods of its life (be it at two different spring periods or at two stages of its age: ram lamb and adult). Eighteen Ile-de-France ram lambs born in February 1980 and issued from five adult rams whose seasonal variations in TD and AS had been followed for two consecutive years were used in this study. Semen was collected (artificial vagina, 1 ej/male/wk) from October 1980 until December 1983 (slaughter of the animals) every semester during "Periods" (n = 7). Within a same year, the first semester (February to June) was called "Spring", the second (August to December) "Autumn". AS (150 cells/smear) were controlled on each ejaculate and the same week maximum TD of both testes was measured using a calliper. Correlations were calculated for TD and AS between individual values (simple correlations) or between groups of individual values (multiple or canonic correlations) at different periods. These values were chosen as follows: TD: 4th and 5th control for the first period (P1, ram lamb); the three lowest values recorded in April (P2, P4, P6); the three mean highest values recorded in Autumn (P3, P5, P7). AS: the first five measurements in P1; the four highest percentages obtained in March-April in P2, P4, P6 (correlations were not calculated in autumn since AS were very few). 1 a: On the whole, TD increased with the age of the animal until 24-27 months and passed alternatively by minimal (February to April) and maximal (September) values each year. Testis growth due to the effect of photoperiodism started every year in the first days of June. 1 b: Except alterations of semen quality in August 1983 (due to high local temperatures), AS were always higher in Spring than in Autumn, maximal values being reached in February (P4) or in March-April (P2 and P6). Their seasonal variations were thus opposed to those of TD (r = -0,671 between weekly means of AS and TD of the whole group of animals from P2 to P6). II-TD: Individual TD of ram lambs were significantly correlated with those recorded in the same adult animal (P1-P2, P1-P6, but P1-P4 NS) especially in autumn (P1-P3, P1-P5, P1-P7.

Lamb production using superovulation, embryo bisection, and transfer.

In this study, 39 embryos from donor ewes superovulated with follicle stimulating hormone-pituitary (FSH-P) were bisected to produce pairs of monozygotic twin lambs for experimentation. Each pair obtained by bisecting 8-, 9- or 10-day-old embryos was immediately transferred surgically into a recipient ewe at the same physiological stage. Of the 39 recipients which received a pair of half-embryos by transfer into the uterine horn ipsilateral to the corpus luteum, 28 (72%) lambed. Eighteen of 28 recipients lambing (64%) produced pairs, i.e., 7 male and 11 female pairs. Ten of 28 lambings produced a single lamb, i.e., six males and four females. Overall yield (the number of lambs produced in relation to the number of embryos used) was 118%. This percentage tended to increase, depending on the day of collection (Day 8, 100%; Day 9, 118%; and Day 10, 131%).

Transfer of superovulated sheep embryos obtained with different FSH-P.

Embryo transfer is one way of accelerating genetic improvement in sheep. One of the main obstacles has been the production of good-quality embryos. The use of progestagens and the stimulation of ovulation with follicle stimulating hormone pituitary extract (FSH-P) has permitted the superovulation of donor and recipient ewes and the synchronization of their cycles. The injection of 16 mg FSH-P at the end of progestin treatment gave means of 9 +/- 1.5, 12 +/- 1.5, and 19.5 +/- 2.6 corpora lutea per ewes in the Préalpes, Lacaune, and Romanov x Préalpes breeds respectively (this last breed is particularly prolific). Twenty Préalpes donor ewes produced 133 embryos that were recovered surgically at Day 6 of gestation; of these, 99 morulae were transferable. Forty-five morulae transferred surgically into 24 Préalpes recipient ewes yielded 16 pregnant ewes and 27 lambs (1.7 per ewe). Twenty-two Lacaune ewes yielded 204 embryos, of which 152 morulae were transferable. Of 76 recipients, 58 became pregnant and gave birth to 97 lambs (1.7 per ewe). During anoestrus, the mean ovulation rate decreased from 11.2 to 8.4; 40.6% of the embryos recovered were of transferable quality versus 74.5% during the normal breeding season. An improved superovulation technique, based on the use of FSH-P with a known follicle stimulating hormone to luteinizing hormonal (FSH/LH) ratio, provided us with good-quality embryos. This treatment must be adapted to the season.

[Seasonal variation in the testis diameter and sperm morphology in the Vendean ram and Texel ram]. / Variations saisonnières du diamètre testiculaire et de la morphologie des spermatozoïdes chez le bélier Vendéen et chez le bélier Texel.

Nineteen Vendean (V) rams (7 yearlings about 15 months old and 12 adults of greater than 2 years) and 13 Texel (T) adult (greater than 2 years) rams were controlled for body weight (BW), testis diameter (TD) and sperm morphology for two consecutive years (1982 and 1983) from February to June (T) or July (V) and from September to November the first year, from February to June and from September to October the second year. BW was recorded every month. TD was measured once weekly using a caliper. The percentage of abnormal spermatozoa (MA) was determined on each ejaculate (1 ej/ram/week) as described previously (Colas, 1980). BW increased rapidly (P less than 0.01) in the T rams the first 4 months and did not augment significantly any more afterwards. In the V breed, no significant variations appeared at any time of the experiment, except in the last month when BW decreased (P less than 0.05). However, in the yearlings (V), there was considerable body growth between February and October 1982 but no significant growth afterwards. In the adults, no statistical variations occurred at any time of the experimental period. TD varied greatly during the year in the V rams. During both years, it was minimal in May (59.0 +/- 5.7 m/m in 1982; 58.1 +/- 4.4 m/m in 1983); in September it was already regressing. There were also large seasonal variations in the TD profile of the T breed but its curve was different from one year to another: the mean value was minimal in February 1982 59.5 +/- 6.0 m/m) and from February (67.5 +/- 4.4 m/m) to May (68.6 +/- 5.0 m/m 1983. In both breeds, testicular growth always started in June, i.e. during the long days, whatever the year. The importance of the variations and their time of onset during the year suggest that both breeds were very sensitive to photoperiod. These results also permit a generalization of the idea that testicular activity in male sheep recommences in the long days. Both breeds showed large individual variations between the minimal values recorded in spring and the highest ones measured in September (2.7 to 78.0% and 2 to 100%, respectively, for V and T rams in 1982; 5.5 to 100% and 14 to 84% in 1983). On the whole, MA were more frequent from February to June than from September to October or November in both breeds. In the V breed, MA were highest in April (35.1% in 1982, 33.1% in 1983).(ABSTRACT TRUNCATED AT 400 WORDS)

[Influence of the photoperiod on the production and fecundity of spermatozoa in the adult Ile-de-France ram]. / Influence de la durée d'éclairement sur la production et la fécondance des spermatozoïdes chez le bélier adulte Ile-de-France.

Two groups, A and B, of 5 adult Ile-de-France rams each of the same age were exposed for 52 weeks (October 1975 to October 1976) to an artificial light regime reproducing natural variations of daylength (DL). In group A, DL increased from 8 to 16 h during the first 26 weeks and then decreased. In group B, DL varied in an opposite manner. Light intensity was constant (300 lux/m2) and ambient temperature was controlled (20 +/- 4 degrees C). The rams were collected using an artificial vagina twice weekly (2 X 2 ejaculates/male/week) during the whole experimental period (EP). The volume and concentration of each ejaculate were recorded. The pH of the raw semen was also measured on the first ejaculate once a week or every two weeks, according to material possibilities. The unselected ejaculates of all rams were used for artificial insemination (AI) on the same days in the same flocks between January and October 1976. AI (980 ewes) was always carried out after oestrus synchronization (FGA + PMSG). Amongst these ejaculates, some (3 ejaculates/male/group/period) could be used for both in vitro (pH) and in vivo (AI) measurements: - Weekly sperm production (SP) varied in an opposite manner in both groups during the whole EP. In group A, minimal (10.6 +/- 4.0.10(9) and maximal (25.5 +/- 5.1.10(9] levels were reached from weeks 12 to 28 and weeks 38 to 52, respectively. In group B, SP was maximal (21.8 +/- 2.3.10(9] from weeks 9 to 24 and minimal (12.7 +/- 5.2.10(9] from weeks 36 to 49. The differences between rams were significant within periods of high and low production. - The sperm pH was somewhat higher in both groups when DL increased and vice-versa. Fertility after AI was higher with rams exposed to short days than with rams exposed to long days (65.7 vs 56.7%; P less than 0.01). The season of AI had no effect on mean fertility (61.0 vs 62.3% for ewes inseminated from January to June and from July to October, respectively), whatever the DL. No correlation was found between the pH of the sperm and its fertilizing ability. From January to April, a highly significant correlation existed between fertility and prolificacy in the 10 rams (r = + 0.607). These results show that photoperiodism has an important effect on both ejaculated sperm production and fertility in rams.(ABSTRACT TRUNCATED AT 400 WORDS)

[The first cleavage of tubal sheep eggs after fertilization with epididymal or ejaculated spermatozoa]. / Observations sur les premiers clivages des oeufs intratubaires de brebis après fécondation avec des spermatozoïdes épididymaires ou éjaculés.

Prealpes ewes (N = 40) were inseminated with mature (ejaculated or epididymal cauda) or immature (epididymal corpus) spermatozoa deposited in the uterine cavity at the time of an induced ovulation (61 hrs. after PMSG injection). Intratubal eggs, recovered and examined either 38 or 48 hrs. later, showed significant differences in segmentation depending on the degree of epididymal maturation of the spermatozoa. Proportion cleaved: 85% of eggs were cleaved after insemination with mature spermatozoa and only 30% after immature spermatozoa were used. Stage of cleavage: 48 hrs. after insemination with mature spermatozoa, 87% eggs were beyond the 4-cell stage, versus 0% after immature spermatozoa.

[Seasonal variations of sperm quality in adult Ile-de-France rams. II. Fertilizing ability and its relation to qualitative criteria in vitro]. / Variations saisonnières de la qualité du sperme chez le bélier Ile-de-France. II. Fécondance: relation avec les critères qualitatifs observés in vitro.

Six adult Ile-de-France rams of different origins, registered in a Flock book, were used in AI for three consecutive years on the same flock (IF) in March-April and in September. The animals (males and females) were always kept in a sheep-fold (natural daylight conditions). They received the same feed throughout the whole experiment. Using the unselected ejaculate(s) of each ram (1 dose diluted sperm/female), artificial inseminations were carried out after oestrus synchronization (FGA + PMSG). Massal motility, percentage of nigrosin-eosin-stained spermatozoa and percentage of morphologically abnormal sperm were determined (150 cells/smear) on each ejaculate (or pooled ejaculates of each ram) used for insemination. 1.--In the spring, sperm fertility was strongly diminished (47.1 vs 68.4 p. 100 LR; P less than 0.01, respectively, in spring and autumn), and the differences between the rams were significant (31.7 to 70.3 p. 100; 0.01 less than P less than 0.02 in spring vs 60.0 to 80.0; P greater than 0.05 in autumn). 2.--Fertilizing ability was related neither to massal motility nor to the proportion of stained cells, although, on the whole, the latter was somewhat higher in spring than in autumn. In the spring, there was a close relationship between the fertility of the ejaculate and its proportion of abnormalities (r = -0.83; P less than 0.01). Fertility was also related to the percentage of abnormal heads (r = -0.59; P less than 0.05). In autumn, when the proportion of total abnormalities was much lower and almost exclusively made up of slight tail damages, this relationship disappeared. Morphological examination of the semen is thus a good fertility test. These results show the importance of the ram in successful breeding in an anoestrous period. Morphological test in the spring must be used to detect those males which are most affected by increasing daylight since, at that time (March-April), a ram maintains nearly the same morphological profile from one year to the next (Colas, 1980).

Biochemical and physiological studies of androgen-binding protein in the reproductive tract of the ram.

The electrophoretic mobility, effect of pronase, temperature stability, affinity constant and specificity of androgen-binding protein (ABP) were compared in rete testis fluid (RTF), cauda epididymal plasma (CEP) and seminal plasma (SP) of the ram in which the levels of ABP, dihydrotestosterone (DHT), total protein and the number of spermatozoa were also measured. The characteristics of the ABP appeared to be almost identical in all 3 fluids. ABP was highly concentrated in the cauda epididymidis although 50-75% of it was utilized or destroyed during transit through the epididymis. The levels of ABP were higher in the breeding season and positively correlated with DHT in RTF and SP. It is concluded that ABP might be responsible for the increase in DHT in the reproductive tract of the ram during the breeding season and that ABP in the SP might serve as a useful marker of Sertoli cell function in the ram.

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.